Mutants of streptococcal toxin A and methods of use

ABSTRACT

This invention is directed to mutant SPE-A toxins or fragments thereof, vaccine and pharmaceutical compositions, and methods of using the vaccine and pharmaceutical compositions. The preferred SPE-A toxin has at least one amino acid change and is substantially non-lethal compared with the wild type SPE-A toxin. The mutant SPE-A toxins can form vaccine compositions useful to protect animals against the biological activities of wild type SPE-A toxin.

BACKGROUND OF THE INVENTION

Streptococcus pyogenes, also known as β-hemolytic group A streptococci(GAS) is a pathogen of humans which can cause mild infections such aspharyngitis and impetigo. Post infection autoimmune complications canoccur, namely rheumatic fever and acute glomerulonephritis. GAS alsocauses severe acute diseases such as scarlet fever and streptococcaltoxic shock syndrome (STSS). Severe GAS infections were a large problemin the U.S. and throughout the world at the beginning of this century.In the mid-forties, the number of cases and their severity decreasedsteadily for yet not completely understood reasons. However, morerecently, a resurgence of serious diseases caused by GAS has been seensuch that there may be 10-20,000 cases of STSS each year in the UnitedStates. As many as 50 to 60% of these patients will have necrotizingfascitis and myositis; 30 to 60% will die and as many as one-half of thesurvivors will have limbs amputated.

In 1986 and 1987 two reports described an outbreak of severe GASinfections localized in the Rocky Mountain area. These reports have beenfollowed in the past few years by several others describing a diseasewith analogous clinical presentation. The symptoms described for thisdisease were very similar to those described for toxic shock syndrome(TSS), and in 1992 a committee of scientists gave to this clinicalpresentation the formal name of STSS, and set the criteria for itsdiagnosis. STSS is defined by the presence of the following:

-   -   1. hypotension and shock;    -   2. isolation of group A streptococci;    -   3. two or more of the following symptoms: fever 38.5° C. or        higher, scarlet fever rash, vomiting and diarrhea, liver and        renal dysfunction, adult respiratory distress syndrome, diffuse        intravascular coagulation, necrotizing fascitis and/or myositis,        bacteremia.

Streptococcal isolates from STSS patients are predominantly of M type 1and 3, with M18 and nontypable organisms making up most of the reminder.The majority of M1, 3, 18, and nontypable organisms associated with STSSmake pyrogenic exotoxin A (SPE-A, scarlet fever toxin A). In contrast,only 15% of general streptococcal isolates produce this toxin. Moreover,administration of SPE-A to a rabbit animal model and in two accidentalhuman inoculations can reproduce the symptoms of STSS.

SPE-A is a single peptide of molecular weight equal to 25,787 daltons,whose coding sequence is carried on the temperate bacteriophage T12.speA, the gene for SPE-A, has been successfully cloned and expressed inEscherichia coli. SPE-A is a member of a large family of exotoxinsproduced by streptococci and staphylococci, referred to as pyrogenictoxins based upon their ability to induce fever and enhance hostsusceptibility up to 100,000 fold to endotoxin.

Recently these toxins have been referred to as superantigens because oftheir ability to induce massive proliferation of T lymphocytes,regardless of their antigenic specificity, and in a fashion dependent onthe composition of the variable part of the β chain of the T cellreceptor. These toxins also stimulate massive release of IFN-γ, IL-1,TNF-α and TNF-β. Other members of this family are streptococcalpyrogenic exotoxins type B and C, staphylococcal toxic shock syndrometoxin 1, staphylococcal enteroxtoxins A, B, Cn, D, E, G and H, andnon-group A streptococcal pyrogenic exotoxins. These toxins have similarbiochemical properties, biological activities and various degrees ofsequence similarity.

The most severe manifestations of STSS are hypotension and shock, thatlead to death. It is generally believed that leakage of fluid from theintravascular to the interstitial space is the final cause ofhypotension, supported by the observation that fluid replacement therapyis successful in preventing shock in the rabbit model of STSS describedabove. It has been hypothesized that SPE-A may act in several ways onthe host to induce this pathology.

SPE-A has been shown to block liver clearance of endotoxin of endogenousflora's origin, by compromising the activity of liver Kuppfer cells.This appears to cause a significant increase in circulating endotoxin,that through binding to lipopolysaccharide binding protein (LBP) andCD14 signaling leads to macrophage activation with subsequent release ofTNF-α and other cytokines. Support for the role of endotoxin in thedisease is given by the finding that the lethal effects of SPE-A can beat least partially neutralized by the administration to animals ofpolymyxin B or by the use of pathogen free rabbits.

Another modality of induction of shock could be the direct activity ofthe toxin on capillary endothelial cells. This hypothesis stems from thefinding that the staphylococcal pyrogenic toxin TSST-1 binds directly tohuman umbilical cord vein cells and is cytotoxic to isolated porcineaortic endothelial cells.

Another of the toxin's modality of action includes itssuperantigenicity, in which the toxin interacts with and activates up to50% of the host T lymphocytes. This massive T cell stimulation resultsin an abnormally high level of circulating cytokines TNF-β andIFN-(which have direct effects on macrophages to induce release of TNF-αand IL-1. These cytokines may also be induced directly from macrophagesby SPE-A through MHC class II binding and signalling in the absence of Tcells. The elevated levels of TNF-α and -β cause several effectstypically found in Gram negative induced shock, among which is damage toendothelial cells and capillary leak. However, the administration toSPE-A treated rabbits of cyclosporin A, which blocks upregulation ofIL-2 and T cell proliferation, did not protect the animals from shock,suggesting that additional mechanisms may be more important in causingcapillary leak.

Thus, there is a need to localize sites on the SPE-A moleculeresponsible for different biological activities. There is a need todevelop variants of SPE-A that have changes in biological activitiessuch as toxicity and mitogenicity. There is a need to developcompositions useful in vaccines to prevent or ameliorate streptococcaltoxic shock syndrome. There is also a need to develop therapeutic agentsuseful in the treatment of streptococcal toxic shock syndrome and otherdiseases.

SUMMARY OF THE INVENTION

This invention includes mutant SPE-A toxins and fragments thereof,vaccines and pharmaceutical compositions and methods of using vaccinesand pharmaceutical compositions.

Mutant SPE-A toxins have at least one amino acid change and aresubstantially nonlethal as compared with a protein substantiallycorresponding to a wild type SPE-A toxin. For vaccine compositions,mutant toxins also stimulate a protective immune response to at leastone biological activity of a wild type SPE-A toxin. Mutant toxins forvaccine compositions are optionally further selected to have a decreasein enhancement of endotoxin shock and a decrease in T cell mitogenicitywhen compared to the wild type SPE-A. An especially preferred mutant forvaccine compositions is one that has a change at an amino acidequivalent to amino acid 20 of a wild type SPE-A toxin. Forpharmaceutical compositions, it is preferred that a mutant toxin issubstantially nonlethal while maintaining T cell mitogenicity comparableto a wild type SPE-A toxin.

The invention also includes fragments of a wild type speA toxin andmutants of speA toxins. Fragments and peptides derived from wild typeSPE-A are mutant SPE-A toxins. Fragments can include different domainsor regions of the molecule joined together. A fragment is substantiallynonlethal when compared to a wild type SPE-A toxin. For mutant toxins, afragment has at least one amino acid change compared to a wild typeSPE-A amino acid sequence. Fragments are also useful in vaccine andpharmaceutical compositions.

The invention also includes expression cassettes, vectors andtransformed cells. An expression cassette comprises a DNA sequenceencoding a mutant SPE-A toxin or fragment thereof operably linked to apromoter functional in a host cell. DNA cassettes are preferablyinserted into a vector. Vectors include plasmids or viruses. Vectors areuseful to provide template DNA to generate DNA encoding a mutant SPE-Atoxin. DNA cassettes and vectors are also useful in vaccinecompositions. Nucleic acids encoding a mutant SPE-A toxin or fragmentthereof can be delivered directly for expression in mammalian cells. Thepromoter is preferably a promoter functional in a mammalian cell.Nucleic acids delivered directly to cells can provide for expression ofthe mutant SPE-A toxin in an individual so that a protective immuneresponse can be generated to at least one biological activity of a wildtype SPE-A toxin.

Additional vaccine compositions include stably transformed cells orviral vectors including an expression cassette encoding a mutant SPE-Atoxin or fragment thereof. Viral vectors such as vaccinia can be used toimmunize humans to generate a protective immune response against atleast one biological activity of a wild type SPE-A toxin. Transformedcells are preferably microorganisms such as S. aureus, E. coli, orSalmonella species spp. Transformed microorganisms either include mutantSPE-A toxin or fragment thereof on their surface or are capable ofsecreting the mutant toxin. Transformed microorganisms can beadministered as live, attenuated or heat killed vaccines.

The invention also includes methods of using vaccines and pharmaceuticalcompositions. Vaccines are administered to an animal in an amounteffective to generate a protective immune response to at least onebiological activity of a wild type SPE-A toxin. Preferably, the vaccinecompositions are administered to humans and protect against thedevelopment of STSS. Pharmaceutical compositions are used in methods ofstimulating T cell proliferation. The pharmaceutical compositions areespecially useful in the treatment of cancers that are treated withinterleukins, interferons or other immunomodulators, T cell lymphomas,ovarian and uterine cancers. A pharmaceutical composition isadministered to a patient having cancer.

The mutant SPE-A toxins and/or fragments thereof and other vaccinecompositions can be useful to generate a passive immune serum. MutantSPE-A toxins or fragments thereof, DNA expression cassettes or vectors,or transformed microorganisms can be used to immunize an animal toproduce neutralizing antibodies to at least one biological activity ofwild type SPE-A. The neutralizing antibodies immunoreact with a mutantSPE-A toxin and/or fragment thereof and the wild type SPE-A toxin.Passive immune serum can be administered to an animal with symptoms of Astreptococcal infection and STSS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Ribbon drawing of the modeled 3-dimensional structure ofstreptococcal pyrogenic exotoxin A. Domain A and B are indicated.

FIG. 2 View of SPE-A as seen from the back in reference to the standardview seen in FIG. 1. Numbered residues are those homologous to residuesin TSST-1 evaluated for reduced systemic lethality.

FIG. 3 shows the DNA sequence (SEQ ID NO:12) and predicted amino acidsequence (SEQ ID NO:13) of the cloned SPE-A toxin from T12.

FIG. 4 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microtiter plates in quadruplicate with SPE-A, K16N-SPE-A, andN20D-SPE-A for 72 hours. Cells were pulsed with [3H] thymidine for 18 to24 hours, harvested onto filters, and [3H] thymidine incorporation wasmeasured in a scintillation counter. Results are expressed as counts perminute (CPM) versus concentrations of toxin in μg/ml. Data presented arefrom the most representative of three independent experiments.

FIG. 5 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microtiter plates in quadruplicate with SPE-A, C87S-SPE-A,C98S-SPE-A, and C90S-SPE-A for 72 hours. Cells were pulsed with [3H]thymidine for 18 to 24 hours, harvested onto filters, and [3H] thymidineincorporation was measured in a scintillation counter. Results areexpressed as counts per minute (CPM) versus concentrations of toxin inμg/ml. Data presented are from the most representative of threeindependent experiments.

FIG. 6 T cell proliferation assay. Rabbit splenocytes were incubated in96 well microtiter plates in quadruplicate with SPE-A, K157E-SPE-A, andS195A-SPE-A for 72 hours. Cells were pulsed with [3H] thymidine for 18to 24 hours, harvested onto filters, and [3H] thymidine incorporationwas measured in a scintillation counter. Results are expressed as countsper minute (CPM) versus concentrations of toxin in μg/ml. Data presentedare from the more representative of three independent experiments.

FIG. 7. Superantigenicity of wild type SPE A compared to single mutant.Rabbit spleen cells were incubated for 4 days with SPE A or mutants atthe indicated doses. Four replicate wells were used at each dose of SPEA and mutants. On day 3, 1 μCI 3H thymidine was added to each well.Superantigenicity index=3H thymidine incorporation by splenocytes in thepresence of SPE A or mutants divided by 3H thymidine incorporation inthe absence of SPE A or mutants.

FIG. 8. Superantigenicity of wild type SPE A compared to double mutants.Methods used were those described in FIG. 7.

FIG. 9. SPE A Inhibition by Immunized Rabbit Sera. Rabbit sera fromrabbits immunized with single and double mutants was used to demonstratethe ability of the sera to neutralize splenocyte mitogenicity in thepresence of SPE A.

FIG. 10 shows a front view of a ribbon structure of SPE-A oriented toshow locations contacting major histocompatibility complex type II in acomplex.

FIG. 11 shows a front view of a ribbon diagram of SPE-A oriented to showlocations that contact the T cell receptor in a complex.

FIG. 12 shows a rear view of a ribbon structure of SPE-A oriented toshow residues of the central α helix that form the floor of the groovethat contacts the liver renal tubular cell receptor in a complex withthis receptor.

FIG. 13 shows a front (standard) view of a ribbon diagram of the modeled3-dimensional structure of SPE A. Organized structures such as β-strandsand α-helices are represented. Domains A and B are indicated. Alphacarbons of the mutated residues and certain other residues arerepresented as spheres.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to mutant SPE-A toxins and fragments thereof,vaccine and pharmaceutical compositions including mutant SPE A toxins orfragments thereof, methods of preparing mutant SPE-A toxins andfragments thereof, and methods of using SPE-A toxins and fragmentsthereof.

Mutant SPE-A toxins are proteins that have at least one amino acidchange and have at least one change in a biological function comparedwith a protein substantially corresponding to a wild type SPE-A toxin.Preferably, the mutant SPE-A toxin is substantially nonlethal whencompared to a wild type SPE-A toxin at the same dose. Mutant SPE-Atoxins can be generated using a variety of methods includingsite-directed mutagenesis, random mutagenesis, conventional mutagenesis,in vitro mutagenesis, spontaneous mutagenesis and chemical synthesis.Mutant SPE-A toxins are preferably selected to: 1) ensure at least onechange in an amino acid; and 2) to have a change in at least onebiological function of the molecule preferably a decrease or eliminationof systemic lethality. The mutant toxins are useful in vaccinecompositions for protection against at least one biological activity ofSPE-A toxin such as prevention or amelioration of STSS, in methods oftreating animals with symptoms of STSS, and in methods for stimulating Tcell proliferation and in the treatment of cancer. Single, double, andtriple SPE-A mutants were tested and the antibody to the mutantsinhibited cell responses to SPE A.

A. Mutant SPE-A Toxins or Fragments Thereof, Vaccine and PharmaceuticalCompositions

The invention includes mutant SPE-A toxins that have at least one aminoacid change and that have at least one change in a biological activitycompared with proteins that substantially correspond to and have thesame biological activities as wild type SPE-A.

Wild type SPE-A toxin is encoded by a gene speA found on bacteriophageT12. The wild type SPE-A toxin has a molecular weight of 25,787 Daltonsas calculated from the deduced amino acid sequence of the matureprotein. A DNA sequence encoding a wild type SPE-A toxin and thepredicted amino acid sequence for a wild type SPE-A toxin is shown inFIG. 3. A DNA sequence encoding a wild type SPE-A toxin has been clonedin E. coli and S. aureus. Amino acid number designations in thisapplication are made by reference to the sequence of FIG. 3 withglutamine at position 31 designated as the first amino acid. The first30 amino acids represent a leader sequence not present in the matureprotein.

A structural model of a wild type SPE-A toxin is shown in FIG. 1. Thestructural model was constructed by homology modeling usingInsight/Homology program available from BioSym Corp., San Diego, Calif.The model indicates that the wild type SPE-A toxin has several distinctstructural features. These structural features include: helix 2 (aminoacids 11-15); N-terminal alpha helix 3 (amino acids 18-26); helix 4(amino acids 64-72); central-α helix 5 (amino acids 142-158); helix 6(amino acids 193-202); Domain B beta strands including strand 1 (aminoacids 30-36), strand 2 (amino acids 44-52), strand 3 (amino acids55-62), strand 4 (amino acids 75-83), strand 5 (amino acids 95-106);Domain A beta strands including strand 6 (amino acids 117-126), strand 7(amino acids 129-135), strand 8 (amino acids 169-175), strand 9 (aminoacids 180-186), and strand 10 (amino acids 213-220). In addition,cysteine residues at residues 87, 90, and 98 may be important information of putative disulfide bonds or maintaining local 3-Dconformation.

The wild type SPE-A toxin has several biological activities. Thesebiological activities include: 1) fever; 2) STSS; 3) systemic lethalitydue to development of STSS or enhancement of endotoxin shock; 4)enhancing endotoxin shock; 5) induction of capillary leak andhypotension; 6) inducing release of cytokines such as IFN g, IL-1, TNF-αand TNF-β; 7) binding to porcine aortic endothelial cells; 8) binding toMHC class II molecules; 9) binding to T-cell receptors; and 10) T-cellmitogenicity (superantigenicity). These activities can be assayed andcharacterized by methods known to those of skill in the art.

As used herein, the definition of a wild type SPE-A toxin includesvariants of a wild type SPE-A toxin that have the same biologicalactivity of wild type SPE-A toxin. These SPE-A toxins may have adifferent amino acid or their genes may have a different nucleotidesequence from that shown in FIG. 3 but do not have different biologicalactivities. Changes in amino acid sequence are phenotypically silent.Preferably, these toxin molecules have systemic lethality and enhanceendotoxin shock to the same degree as wild type SPE-A toxin shown inFIG. 3. Preferably these toxins have at least 60-99% homology with wildtype SPE-A toxin amino acid sequence as shown in FIG. 3 as determinedusing the SS2 Alignment Algorithm as described by Altschul, S. F., Bull.Math. Bio. 48:603 (1986). Proteins that have these characteristicssubstantially correspond to a wild type SPE A.

A mutant SPE-A toxin is a toxin that has at least one change in a aminoacid compared with a protein substantially corresponding to a wild typeSPE-A toxin. The change can be an amino acid substitution, deletion, oraddition. There can be more than one change in the amino acid sequence,preferably 1 to 6 changes. It is preferred that there are more than onechange in the amino acid sequence to minimize the reversion of mutantSPE-A toxin to the wild type SPE-A toxin having systemic lethality ortoxicity. For mutant SPE-A toxins useful in vaccines, it is preferredthat the change in the amino acid sequence of the toxin does not resultin a change of the toxin's ability to stimulate an antibody responsethat can neutralize wild type SPE-A toxin. For mutant SPE-A toxinsuseful in vaccines, it is especially preferred that the mutant toxinsare recognized by polyclonal neutralizing antibodies to SPE-A toxin suchas from Toxin Technologies in Boca Raton, Fla. or Dr. Schlievert(University of Minnesota, Minneapolis, Minn.) and that the proteolyticprofile is not altered compared with wild type SPE-A.

The changes in the amino acid sequence can be site-specific changes atone or more selected amino acid residues of a wild type SPE-A toxin.Site-specific changes are selected by identifying residues in particulardomains of the molecule as described previously or at locations wherecysteine residues are located. Site-specific changes at a particularlocation can optionally be further selected by determining whether anamino acid at a location or within a domain is identical to or hassimilar properties to an equivalent residue in other homologousmolecules by comparison of primary sequence homology or 3-Dconformation. A homologous molecule is one that can be identified bycomparison of primary sequence homology using the SS2 alignmentalgorithm of Altschul et al., cited supra or a homology modeling programsuch as Insight/Homology from BioSym, San Diego, Calif. A homologousmolecule is one that displays a significant number, typically 30-99%, ofidentical or conservatively changed amino acids or has a similar threedimensional structure, typically RMS error for conserved regions of <2Angstroms, when compared to a wild type SPE-A toxin.

Changes in the amino acid sequence at a particular site can be randomlymade or specific changes can be selected. Once a specific site isselected it is referred to by its amino acid number designation and bythe amino acid found at that site in the wild type SPE-A as shown inFIG. 3. The amino acid number designations made in this application areby reference to the sequence in FIG. 3 with the glutamine at position 31being counted as the first amino acid. Equivalent amino acidscorresponding to those identified at a particular site in proteinssubstantially corresponding to a wild type SPE-A toxin may havedifferent amino acid numbers depending on the reference sequence or ifthey are fragments. Equivalent residues are also those found inhomologous molecules that can be identified as equivalent to amino acidsin proteins substantially corresponding to a wild type SPE-A toxineither by comparison of primary amino acid structure or by comparison toa modeled structure as shown in FIG. 1 or by comparison to a knowncrystal structure of a homologous molecule. It is intended that theinvention cover changes to equivalent amino acids at the same or similarlocations regardless of their amino acid number designation.

If a specific substitution is selected for an amino acid at a specificsite, the amino acid to be substituted at that location is selected toinclude a structural change that can affect biological activity comparedwith the amino acid at that location in the wild type SPE-A. Thesubstitution may be conservative or nonconservative. Substitutions thatresult in a structural change that can affect biological activityinclude: 1) change from one type of charge to another; 2) change fromcharge to noncharged; 3) change in cysteine residues and formation ofdisulfide bonds; 4) change from hydrophobic to hydrophilic residues orhydrophilic to hydrophobic residues; 5) change in size of the aminoacids; 6) change to a conformationally restrictive amino acid or analog;and 7) change to a non-naturally occurring amino acid or analog. Thespecific substitution selected may also depend on the location of thesite selected. For example, it is preferred that amino acids in theN-terminal alpha helix have hydroxyl groups to interact with exposedamide nitrogens or that they be negatively charged to interact with thepartial positive charge present at the N-terminus of the α helix.

Mutant toxins may also include random mutations targeted to a specificsite or sites. Once a site is selected, mutants can be generated havingeach of the other 19 amino acids substituted at that site using methodssuch as described by Aiyar et al., Biotechniques 14:366 (1993) or Ho etal. Gene 77:51-54 (1984). In vitro mutagenesis can also be utilized tosubstitute each one of the other 19 amino acids or non-naturallyoccurring amino acids or analogs at a particular location using a methodsuch as described by Anthony-Cahill et al., Trends Biochem. Sci. 14:400(1989).

Mutant toxins also include toxins that have changes at one or more sitesof the molecule not specifically selected and that have a change inamino acids that is also not specifically selected but can be any one ofthe other 19 amino acids or a non-naturally occurring amino acid.

Substitutions at a specific site can also include but are not limited tosubstitutions with non-naturally occurring amino acids such as3-hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid,2-aminopimilic acid, ornithine, homoarginine, N-methyllysine, dimethyllysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutryicacid, hydroxylysine, substituted phenylalanine, norleucine, norvaline,g-valine and halogenated tyrosines. Substitutions at a specific site canalso include the use of analogs which use non-peptide chemistryincluding but not limited to ester, ether and phosphoryl and boronlinkages.

The mutant toxins can be generated using a variety of methods. Thosemethods include site-specific mutagenesis, mutagenesis methods usingchemicals such as EMS, or sodium bisulfite or UV irradiation, byspontaneous mutation, by in vitro mutagenesis and chemical synthesis.Methods of mutagenesis can be found in Sambrook et al., A Guide toMolecular Cloning, Cold Spring Harvard, N.Y. (1989). The especiallypreferred method for site-specific mutagenesis is using asymmetric PCRwith three primers as described by Perrin and Gilliland, 1990. NucleicAcid Res. 18:7433.

Once a mutant SPE-A toxin is generated having at least one amino acidchange compared with a protein substantially corresponding to the wildtype SPE-A toxin, the mutant SPE-A toxin is screened for nonlethality.It is preferred that mutant SPE-A toxins selected from this screeningare substantially nonlethal in rabbits when administered using aminiosmotic pump (as described in Example 2) at the same dose or agreater dose than a wild type SPE-A toxin. A mutant SPE-A toxin orfragment thereof is substantially nonlethal if when administered to arabbit at the same dose as the wild type toxin less than about 10-20% ofrabbits die. Nonlethal mutant toxins are useful in vaccine andpharmaceutical compositions. While not meant to limit the invention, itis believed that some amino acid residues or domains that affectsystemic lethality are separable from other biological activitiesespecially T cell mitogenicity.

For mutant toxins useful in vaccine composition it is further preferredthat the mutant SPE-A toxins are screened for those that can stimulatean antibody response that neutralizes wild type SPE-A toxin activity. Amethod for selecting mutant toxins that can stimulate an antibodyresponse that neutralizes wild type SPE-A toxin activity is to determinewhether the mutant toxin immunoreacts with polyclonal neutralizingantibodies to wild type SPE-A such as available from Toxin Technologies,Boca Raton, Fla. or Dr. Schlievert. Methods of determining whethermutant SPE-A toxins immunoreact with antibodies to wild type SPE-A toxininclude ELISA, Western Blot, Double Immunodiffusion Assay and the like.

Optionally, the mutant toxins can also be screened to determine if theproteolytic profile of the mutant toxin is the same as the wild typeSPE-A toxin. In some cases, it is preferred that the mutants generateddo not substantially change the overall three-dimensional conformationof the mutant toxin compared with the wild type SPE-A toxin. One way ofexamining whether there has been a change in overall conformation is tolook at immunoreactivity of antibodies to wild type SPE-A toxin and/orto examine the proteolytic profile of mutant SPE-A toxins. Theproteolytic profile can be determined using such enzymes as trypsin,chymotrypsin, papain, pepsin, subtilisin and V8 protease in methodsknown to those of skill in the art. The proteolytic profile of wild typeSPE-A with the sequence shown in FIG. 3 is known. The mutants that havea similar profile to that of wild type SPE-A are selected.

Optionally, mutant toxins can also be screened and selected to haveother changes in biological activity. As described previously, there areseveral biological activities associated with wild type SPE-A toxin.Those biological activities include: 1) fever; 2) STSS; 4) enhancementof endotoxin shock; 5) capillary leak and hypotension; 6) inducingrelease of cytokines such as IFN gamma, IL-1, TNF-α and TNF-β; 7)binding to endothelial cells; 8) binding to MHC class II molecules; 9)binding to T-cell receptors; and 10) T-cell mitogenicity(superantigenicity). These biological activities can be measured usingmethods known to those of skill in the art.

For mutant SPE-A toxins or fragments thereof useful in vaccinecompositions, it is preferred that they are substantially nontoxic andimmunoreactive with neutralizing antibodies to wild type SPE-A.Neutralizing antibodies include those that inhibit the lethality of thewild type toxin when tested in animals. Optionally, mutant SPE-A toxinscan have a change in one or more other biological activities of wildtype SPE-A toxin as described previously.

Optionally, preferred mutant toxins for vaccine compositions are furtherscreened and selected for a lack of potentiation of endotoxin shock. Thepreferred assay for examining a lack of enhancement of endotoxin shockis described in Example 4. Rabbits preferably have no demonstrablebacterial or viral infection before testing. A lack of potentiation ofor substantially no enhancement of endotoxin shock is seen when lessthan about 25% of the animals develop shock when the mutant SPE toxin iscoadministered with endotoxin as compared to wild type SPE-A activity atthe same dose. More preferably, none of the animals develop shock.

Optionally, preferred mutants for vaccine compositions also are furtherscreened and selected for a change in T cell mitogenicity. A change inT-cell mitogenicity can be detected by measuring T-cell proliferation ina standard 3H thymidine assay using rabbit lymphocytes as described inExample 4; by measuring levels of production of cytokines such as IFNgamma or TNF-β; by determining the Vβ type of T cell response or bydetermining the interaction of the molecules with MHC class IIreceptors. The preferred method for detecting a decrease in T-cellmitogenicity is to measure T-cell proliferation of rabbit lymphocytes inthe presence and absence of the mutant toxin. Responses of T cells towild type SPE-A toxin is greatly enhanced above a normal in vitroresponse to an antigen. A substantial decrease in T cell mitogenicity isseen when the mutant SPE-A toxin does not stimulate a T cellproliferative response greater than the stimulation with an antigen ornegative control. Preferably, a decrease is seen such that the T cellproliferation response to the mutant SPE-A toxin is no more thantwo-fold above background when measured using rabbit lymphocytes at thesame dose as the wild type SPE-A toxin.

Optionally, the mutant SPE-A toxins useful in vaccine compositions arefurther screened and selected for a decrease in capillary leak inendothelial cells. The preferred method is using porcine aorticendothelial cells as described by Lee t el., J. Infect. Dis. 164:711(1991). A decrease in capillary leak in the presence of mutant SPE-Atoxins can be determined by measuring a decrease in release of aradioactively labeled compound or by a change in the transport of aradioactively labeled compound. A decrease in capillary leak is seenwhen the release or transport of a radioactively labeled compound isdecreased to less than about two fold above background when comparedwith the activity of a wild type toxin.

The especially preferred mutant SPE-A toxins useful in vaccinecompositions are not biologically active compared with proteins thathave wild type SPE-A toxin activity. By nonbiologically active, it ismeant that the mutant toxin has little or no systemic lethality, haslittle or no enhancement of endotoxin shock and little or no T cellmitogenicity. Preferably, the mutant SPE-A toxins selected for vaccinecompositions substantially lack these biological activities, i.e., theyreact like a negative control or they stimulate a reaction no more thantwo-fold above background.

Changes in other biological activities can be detected as follows.Binding to MHC class II molecules can be detected using such methods asdescribed by Jardetzky, Nature 368:711 (1994). Changes in fever can bedetected by monitoring temperatures over time after administration ofthe mutant SPE-A toxins. Changes in the levels of cytokine production inthe presence of mutant SPE-A toxins can be measured using methods suchas are commercially available and are described by current protocols inimmunology. (Ed. Coligan, Kruisbeck, Margulies, Shevach, and Stroker.National Institutes of Health, John Wiley and Sons, Inc.)

Specific examples of mutant SPE-A toxins that have at least one aminoacid change and that are substantially nontoxic are described.

The especially preferred mutants for vaccine compositions are mutantSPE-A toxins that immunoreact with polyclonal neutralizing antibodies towild type SPE-A toxin, are nontoxic, and optionally have a decrease inpotentiation of endotoxin shock and a decrease in T-cell mitogenicity.The especially preferred mutants have a change in the asparagine atamino acid 20 such as the mutant N20D that has an aspartic acidsubstituted for asparagine at residue 20 in the mature toxin (N20D). TheN20D mutant has been shown to be nontoxic, to have no enhancement ofendotoxin shock and a 5-fold decrease in T cell mitogenicity. Inaddition, changes at amino acid 98 that result in a lack of a cysteinegroup at that location also result in a mutant toxin that has a decreasein enhancement in endotoxin shock and a four-fold decrease inmitogenicity. The especially preferred mutants at this location have aserine substituted for a cysteine (C98S).

The preferred mutants for stimulation of T-cell proliferation and in thetreatment of cancer are those mutant toxins that are substantiallynonlethal. It is preferred that these mutant toxins retain T-cellmitogenicity at least at the level of wild type SPE-A toxin. Theespecially preferred mutants have an amino acid change at residue 157 ofthe wild type SPE-A such as substitution of glutamic acid for lysine atthat residue (K157E). The K157E mutant has been shown to be nonlethalbut retains mitogenicity comparable to the wild type SPE-A toxin.

Mutants can be generated to affect a functional change by changing aminoacids in a particular domain of a molecule as follows. A molecular modelof wild type SPE-A toxin is shown in FIG. 1. The especially preferreddomains include the N-terminal α helix 3 (amino acids 18-26), thecentral α helix 5 (amino acids 142-158), the Domain B beta strands(amino acids 30-36; 44-52; 55-62; 75-83; and 95-106), and the Domain Abeta strands (amino acids 117-126; 129-135; 169-175; 180-186; and213-220). Cysteine residues at positions 87, 90, and 98 may also beimportant.

While not meant to limit the invention, it is believed that thesedomains form specific 3-D conformations that are important in thebiological functions of the wild type SPE-A activity. As can be seen inFIG. 2, the N-terminal α helix and central α helix are closely situatedso that residues here may be especially important in the toxicity ofwild type SPE-A molecules. In addition, amino acids in the bordering Bstrands that are in close proximity to the central alpha helix may alsobe important in toxicity. The molecular models as shown in FIGS. 1 and 2help to identify surface residues and buried residues of the structuraldomains.

For vaccine compositions, changes are preferably made to the residues inN-terminal alpha helix 3 (residues 18-26) are screened and selected todecrease systemic lethality or enhancement of endotoxin or T cellmitogenicity or all three.

A specific example of a change in the N-terminal alpha helix 3 is achange in amino acid at residue 20. A change at this residue fromasparagine to aspartic acid results in a decrease in enhancement ofendotoxin shock, a decrease in systemic lethality, and a five-folddecrease in mitogenicity. Other changes at residue 20 are preferablythose that change the distribution of charge at the surface residues orthat change the interaction of the N-terminal α helix with the central αhelix. Substitutions at amino acid 20 with charged amino acids such asglutamic acid, lysine, arginine are likely to have the same effect.Changes made in this region are preferably those that decrease insystemic lethality due to STSS.

Preferably, changes are also made in the central α helix 5 residues142-158. Mutants in this region having at least one amino acid changeare preferably selected for a decrease systemic lethality due to STSS. Asimilar central α helix identified in other toxin molecules has beenshown to be associated with toxicity. A specific example is a change atresidue 157. Change at this residue from lysine to glutamic acid resultsin a decrease in enhancement of endotoxin shock and systemic lethalitydue to STSS.

However, T-cell mitogenicity is not affected by a change at thisresidue. These results show that toxicity and enhancement of endotoxinshock are separable activities from T cell mitogenicity. For vaccinecompositions, other mutant toxins with changes in this domain areoptionally screened and selected for a decrease in T cell mitogenicity.A change in the type of charge present at amino acid 157 indicates thata substitution of aspartic acid for the lysine is likely to have asimilar effect.

Preferably changes in domain B beta strands including residues 30-36(beta strand 1), residues 44-52 (beta strand 2), residues 55-62 (betastrand 3), residues 75-83 (beta strand 4), and residues 95-106 (betastrand 5) (domain 5) are screened and selected for nonlethality, andoptionally for a decrease in enhancement of endotoxin shock and/or Tcell mitogenicity. Multiple residues that form N-terminal barrel of betasheet in several toxins such as SEB, SEA, TSST-1 have been shown to beimportant for binding to MHC class II molecules. A decrease in MHC classII binding by mutant toxins can also be selected by using assays such asdescribed by Jardetzky et al., cited supra. Changes to these residuesthat would disrupt beta sheet conformation or change the contactresidues with MHC class II molecules, especially those on the concavesurface of the beta barrel, are selected. See FIG. 1. For vaccinecompositions, it is preferred that changes that may change localconformation do not change the immunoreactivity of the mutant toxinswith polyclonal neutralizing antibodies to the wild type SPE-A toxin.

Preferably changes to Domain A beta strands, including residues 117-126(domain beta strand 6), residues 129-135 (domain 7), residues 169-175(domain 8), residues 180-186 (domain 9), and residues 213-220 (domain10), are selected to be nonlethal, have a decrease in endotoxin shock,and/or have a decrease in T cell mitogenicity. Changes that would alterthe beta sheet conformation without changing the immunoreactivity of themutant SPE-A toxin with polyclonal neutralizing antibodies to wild typeSPE-A toxin are preferably selected.

Superpositioning the three-dimensional structures of four staphylococcalsuperantigens (TSST-1, SEA, SEB, and SEC-3) and of SPE-A demonstratedthat these proteins share 18 structurally conserved amino acids (TableA). Using these 18 structurally conserved amino acid residues asreference points allows superpositioning of the structures of these 5proteins with RMS differences at or below 2 angstroms, which issignificant for proteins with minimal amino acid sequence conservation.This superpositioning based on 18 structurally conserved amino acidsallows detailed comparison of the structure of SPE-A with thestaphylococcal superantigens.

The crystal structure of the complex of staphylococcal superantigen SEBand the class II major histocompatibility complex (MHC-II) shows aminoacids on SEB that contact MHC-II, including those listed on Table B.Superposition of the SPE-A structure indicates the location of the aminoacids of SPE-A that contact MHC-II, contact residues or contact areas,in a complex of these two proteins. These locations are shown in FIG. 10as balls.

Specifically, FIG. 10 shows SPE-A 1 with B domain 2 including β-barrel3, which is made up of various strands and loops. Locations 4-7 are onstrand 8. Location 4 is a distance equivalent to about 3 amino acidsfrom the carboxy terminus of strand 8, which is at the junction ofstrand 8 and loop 9, and just below a turn on strand 8, by theorientation of FIG. 10. Location 4 can be occupied by a polar aminoacid, preferably residue Asn-49 of SPE-A. Location 5 is near the centerof strand 4, and can be occupied by a hydrophobic amino acid, preferablyresidue Ile-47 of SPE-A. A distance of about 1 amino acid intervenesbetween locations 5 and 6. Location 6 can be occupied by a hydrophobicamino acid, preferably Leu-41 of SPE-A. Location 7 is at the aminoterminal end of strand 8. Location 7 can be occupied by a hydrophobicamino acid, preferably by Leu-42 of SPE-A.

Locations 10-12 are on strand 13 of β-barrel 3. Location 10 is aboutthree amino acids distant from the junction of loop 9 and strand 13, andthere is a turn between location 10 and that junction. Location 10 canbe occupied by a charged amino acid, preferably Lys-57 of SPE-A.Location 11 is near the center of strand 13, and can be occupied by acharged amino acid, preferably Lys-58 of SPE-A. Location 11 is proximalto location 5. Location 12 is nearest the junction of loop 14 and strand13, but on strand 13, and can be occupied by a charged amino acid,preferably residue Glu-61 of SPE-A. Location 53 is at the junction ofstrand 13 and loop 14. Location 13 can be occupied by a hydrophobicamino acid, preferably Leu-62 of SPE-A.

Locations 15, 16, and 17 are on loop 18. Location 15 is on loop 18 at aposition where loop 18 crosses a plane defined by strands 8 and 13.Location 15 is adjacent to the center turn 26 of alpha helix 25.Location 15 can be occupied by an unchanged or a polar amino acid,preferably Ser-43 of SPE-A. Location 16 is on loop 18 at a point whereloop 18 has risen above strand 8, as shown in FIG. 10. Location 16 canbe occupied by a polar amino acid, preferably His-44 of SPE-A. Location17 is on loop 18 proximal to locations 6 and 5. Location 17 can beoccupied by a polar amino acid, preferably Gln-40 of SPE-A.

Locations 19-21 are on loop 22. These locations are separated by thedistance of about a single amino acid. Locations 19 and 20 areapproximately midway along the length of loop 22. Location 19 can beoccupied by a neutral or polar amino acid, preferably by Cys-90 ofSPE-A. Location 20 can be occupied by a neutral or polar amino acid,preferably Tyr-88 of SPE-A. Location 21 can be occupied by a hydrophobicamino acid, preferably Leu-86 of SPE-A.

Location 23 is on α-helix 24 in the turn nearest the junction of helix24 and loop 18. Location 23 can be occupied by a polar amino acid,preferably His-44 of SPE-A.

The crystal structure of the complex of staphylococcal SEC and theT-cell receptor shows amino acids on SEC 3 that contact the T-cellreceptor including residues listed in Table C. Super position of theSPE-A structure indicates the location of the amino acids of SPE-A thatcontact the T-cell receptor in a complex of these two proteins. Theselocations are shown in FIG. 11 as balls.

Specifically, with reference to FIG. 11, these include locations 10-12described hereinabove. Locations 27-30 are on loop 22. Each location isadjacent to the preceding location. Location 30 is at the junction ofloop 22 and strand 31. Location 27 can be occupied by a polar aminoacid, preferably Asn-92 of SPE-A. Location 28 can be occupied by aneutral amino acid, preferably Ala-93 of SPE-A. Location 29 can beoccupied by a charged amino acid, preferably Glu-94 of SPE-A. Location30 can be occupied by a charged amino acid, preferably Arg-95 of SPE-A.

Location 32 is on strand 9, at about the middle of strand 9. Location 32can be occupied by a polar amino acid, preferably Asn-54 of SPE-A.Location 33 is at the junction of loop 22 and strand 34. Location 33 canbe occupied by a polar amino acid, preferably Tyr-84 of SPE-A. Location35 is on loop 22 adjacent to location 33. Location 35 can be occupied bya polar amino acid, preferably His-85 of SPE-A.

Locations 36-40 are in N-terminal α-helix 41. Location 36 is on loop 42of N-terminal α-helix 41. Location 36 is the distance of one residueremoved from the junction of N-terminal α-helix 41 with loop 44.Locations 37-40 are adjacent locations in loop 43 of N-terminal alphahelix 41. Location 36 can be occupied by a hydrophobic amino acid,preferably Phe-23 of SPE-A. Location 37 can be occupied by a polar aminoacid, preferably Asn-20 of SPE-A. Location 38 can be occupied by a polaramino acid, preferably Gln-19 of SPE-A. Location 39 can be occupied by ahydrophobic amino acid, preferably Leu-18 of SPE-A. Location 40 can beoccupied by a polar amino acid, preferably Asn-17 of SPE-A.

Locations 45 and 46 are in a region of loop 47 proximal to turn 43 ofthe N-terminal α-helix 41. Location 45 can be occupied by a polar aminoacid, preferably Tyr-160 of SPE-A. Location 46 can be occupied by apolar amino acid, preferably Asn-162 of SPE-A. Locations 45 and 46 areseparated by approximately the distance of one amino acid residue.

Interactions between SPE-A and the liver renal tubular receptor includesinteractions with central α-helix 48 shown in FIG. 12. Locations oncentral α-helix 48 important to interaction with the liver receptorinclude locations 49-54. Locations 49-55 define a surface of the centralα-helix 45 that forms the base of a groove in the structure of SPE-A.Locations 49-54 are preferred locations on this surface. Locations 49and 51 can be occupied by polar residues. Locations 50 and 52-54 can beoccupied by charged residues. Preferably location 49 is Asn-156,location 50 is amino acid Asp-55, location 51 is amino acid Tyr-152,location 52 is amino acid Lys-151, location 53 is amino acid Lys-148, orlocation 54 is amino acid Glu-144. More preferred locations are 50, 51,53, and 54, which have the greatest proportion of the location on thesurface defined by locations 49-55. Location 55 is proximal to thejunction of loop 56 and central α-helix 45. Location 55 can be occupiedby a neutral or polar amino acid, preferably by Thr-141 of SPE-A.

Table B lists residues of SEB that interact with class 2 MHC in thecrystal structure of the complex of these two proteins. Superposition ofthe structures of SEC-3, SEA and TSST-1 with the structure of theSEB:MHC-II complex indicates amino acids on these proteins thatcorrespond to the SEB residues that interact with MHC-II, includingresidues on these proteins listed in Table B. Preferred SPE-A mutantsinclude substitution of an SPE-A residue that corresponds to a residuein SEB, SEC-3, SEA or TSST-1 that interacts with MHC-II. These preferredSPE-A residues include the SPE-A residues listed in Table B.Corresponding residues from the different proteins are listed across therows of the table.

Table C lists residues of SEC-3 that interact with the T-cell receptorin the crystal structure of the complex of these two proteins.Superposition of the structures of SEC-3, SEA and TSST-1 with thestructure of the SEB:T-cell receptor complex indicates amino acids onthese proteins that correspond to the SEB residues that interact withT-cell receptor and inlcudes residues listed in Table C. Preferred SPE-Amutants include substitution of an SPE-A residue that corresponds to aresidue in SEB, SEC-3, SEA or TSST-1 that interact with T-cell receptor.These preferred SPE-A residues include the SPE-A residues listed inTable B. Corresponding residues from the different proteins are listedacross the rows of the table.

Preferred mutants of SPE-A have amino acid substitutions in at least oneof the locations or for at least one of the amino acid residues thatinteracts with the T-cell receptor, MHC-II or the liver neutral tubularcell receptor. These amino acid substitutions can be chosen as describedhereinabove to disrupt the interactions. TABLE A PTSAG CONSERVEDRESIDUES TSST-1 SEA SEB SEC-3 SPE-A TYR 13 TYR 30 TYR 28 TYR 28 TYR 25ASP 27 ASP 45 ASP 42 ASP 42 ASP 39 LYS 58 LYS 81 LYS 78 LYS 78 LYS 72VAL 62 VAL 85 VAL 82 VAL 82 VAL 76 ASP 63 ASP 86 ASP 83 ASP 83 ASP 77GLY 87 GLY 110 GLY 117 GLY 114 GLY 102 THR 89 THR 112 THR 119 THR 116THR 104 LYS 121 LYS 147 LYS 152 LYS 151 LYS 137 LYS 122 LYS 148 LYS 153LYS 152 LYS 138 LEU 129 LEU 155 LEU 160 LEU 159 LEU 145 ASP 130 ASP 156ASP 161 ASP 160 ASP 146 ARG 134 ARG 160 ARG 162 ARG 161 ARG 150 LEU 137LEU 163 LEU 168 LEU 167 LEU 153 LEU 143 LEU 169 LEU 171 LEU 170 LEU 159TYR 144 TYR 170 TYR 172 TYR 171 TYR 160 GLY 152 GLY 182 GLY 185 GLY 184GLY 170 ASP 167 ASP 197 ASP 199 ASP 199 ASP 185 ILE 189 ILE 226 ILE 230ILE 230 ILE 214

TABLE B RESIDUES INVOLVED IN CLASS II MHC INTERACTIONS SEB TSST-1 SEASEC-3 SPE-A Gln 43 Asn 28 Gln 46 Lys 43 Gln 40 Phe 44 Ser 29 Phe 47 Phe44 Leu 41 Leu 45 Leu 48 Leu 45 Leu 42 Tyr 46 Leu 30 Gln 49 Ala 46 Ser 43Phe 47 Gly 31 His 50 His 47 His 44 Asp 45 Gln 92 Lys 71 Gln 95 Asn 92Leu 86 Tyr 94 Gln 73 Ala 97 Tyr 94 Tyr 88 Ser 96 Gly 99 Ser 96 Cys 90Met 215 Asn 175 Arg 211 Met 215 Met 199

TABLE C RESIDUES INVOLVED IN TCR INTERACTIONS TSST-1 SEC-3 SEA SPE-A SEBASN 5 GLY 19 THR 21 ASN 17 GLY 19 THR 20 ALA 22 LEU 18 LEV 20 ASP 8 ASN23 ASN 25 ASN 20 ASN 23 ASP 11 TYR 26 GLN 28 PHE 23 VAL 26 ASN 60 ASN 54LYS 70 TYR 90 GLY 93 TYR 84 TYR 90 VAL 91 TYR 94 HIS 85 TYR 91 GLY 102ASN 92 LYS 103 ALA 93 VAL 104 GLU 94 SER 106 LYS 103 ARG 95 ARG 145 PHE176 ASN 171 ASN 162 TYR 175 GLN 210 SER 206 GLN 194 GLN 210

Mutant SPE-A toxins with changes to cysteine residues or introduction ofdisulfide bonds can be selected that have a decrease in lethality, oroptionally a decrease in enhancement of endotoxin shock, and/or adecrease in T cell mitogenicity. A specific example is change at thecysteine residue 98. A change at this residue from cysteine to serineresults in a mutant toxin with a decrease in mitogenicity aboutfour-fold and a decrease in enhancement in endotoxin shock and adecrease in lethality due to STSS. Changes that eliminate the cysteinegroup at residue 98 can effect biological activity in a similar manneras a substitution with serine. Other changes that could be made atresidue 98 include substitution of the other small aliphatic residuessuch as alanine, glycine or threonine. Changes at other cysteineresidues at amino acid residues 90 and 97 result in a decrease inmitogenicity.

Advantageously, mutant SPE-A toxins useful in treatment methods can begenerated that have more than one change in the amino acid sequence. Itwould be desirable to have changes at more than one location to minimizeany chance of reversion to a molecule having toxicity or lethality. Forvaccine compositions, it is desirable that a mutant toxin with multiplechanges can still generate a protective immune response against wildtype SPE-A and/or immunoreact with neutralizing polyclonal antibodies towild type SPE-A. For pharmaceutical compositions, it is preferred thatmutants with multiple changes are substantially nonlethal whilemaintaining mitogenicity for T cells. It is especially preferable tohave about 2 to 6 changes. Examples of such mutants include those withthe N20D mutation including double mutants such as N20D/K157E,N20D/C98S, triple mutants, such as N20D/D45N/C98S, and the like. Doublemutant N20D/C98S has been deposited with the ATCC and has accession no.55821. Double mutant N20D/C98S has been deposited with the ATCC and hasaccession no. 55822. Triple mutant N20D/D45N/C98S has been depositedwith the ATCC and has accession no. 55993.

Double mutants of SPE A may offer advantages over single mutants. Thiswas evaluated in three experiments detailed in Example 6. Results areprovided in FIGS. 7-9. The data indicated that the N20D/C98S mutant hadless toxicity than the single N20D mutant and the double mutantN20D/K157E was intermediate between the other two proteins. All threemutants were significantly less toxic than wild type SPE A. Sera fromrabbits immunized with the single and double mutants inhibitedlymphocyte proliferation in response to nonmutated SPE A toxin.Lymphocyte proliferation is associated with and necessary for fulltoxicity of the toxin.

Animals were immunized against N20D, N20D/C98S, or N20D/K157E, asdescribed in Example 7. Results are provided in Table 9. Animalsimmunized with either double mutant were completely protected from feverand enhanced susceptibility to endotoxin shock.

Triple mutants are also contemplated in this application and in oneembodiment, the SPE-A mutant N20D/C98S/D45N was tested using the methodsand assays of Examples 1-7 and the primers disclosed herein.

It may also be preferable to delete residues at specific sites such asdeletion of amino acid residue 20 asparagine and/or deletion of aminoacid 157 lysine or 98 cysteine. For vaccine compositions, mutants withdeletions would be selected that immunoreact with polyclonalneutralizing antibodies to wild type SPE-A toxin and/or can stimulate aprotective immune response against wild type SPE-A activity.

Mutant toxins of SPE-A are useful to form vaccine compositions. Thepreferred mutants for vaccine compositions have at least one amino acidchange, are nontoxic systemically, and immunoreact with polyclonalneutralizing antibodies to wild type SPE-A. The especially preferredmutants include those mutant SPE-A toxins with a change at amino acid 20such as N20D, N20D/K157E, N20D/C98S, and mutants with a deletion atresidue 20 asparagine.

Mutant toxins are combined with a physiologically acceptable carrier.Physiologically acceptable diluents include physiological salinesolutions, and buffered saline solutions at neutral pH such as phosphatebuffered saline. Other types of physiological carriers include liposomesor polymers and the like. Optionally, the mutant toxin can be combinedwith an adjuvant such as Freund's incomplete adjuvant, Freund's Completeadjuvant, alum, monophosphoryl lipid A, alum phosphate or hydroxide,QS-21 and the like. Optionally, the mutant toxins or fragments thereofcan be combined with immunomodulators such as interleukins, interferonsand the like. Many vaccine formulations are known to those of skill inthe art.

The mutant SPE-A toxin or fragment thereof is added to a vaccineformulation in an amount effective to stimulate a protective immuneresponse in an animal to at least one biological activity of wild typeSPE-A toxin. Generation of a protective immune response can be measuredby the development of antibodies, preferably antibodies that neutralizethe wild type SPE-A toxin. Neutralization of wild type SPE-A toxin canbe measured including by inhibition of lethality due to wild type SPE-Ain animals. In addition, a protective immune response can be detected bymeasuring a decrease in at least one biological activity of wild typeSPE-A toxins such as amelioration or elimination of the symptoms ofenhancement of endotoxin shock or STSS. The amounts of the mutant toxinthat can form a protective immune response are about 0.1 μg to 100 mgper kg of body weight more preferably about 1 μg to about 100 μg/kg bodyweight. About 25 μg/kg of body weight of wild type SPE-A toxin iseffective to induce protective immunity in rabbits.

The vaccine compositions are administered to animals such as rabbits,rodents, horses, and humans. The preferred animal is a human.

The mutant SPE-A toxins are also useful to form pharmaceuticalcompositions. The pharmaceutical compositions are useful in therapeuticsituations where a stimulation of T-cell proliferation may be desirable,such as in the treatment of cancer. The preferred mutant SPE-A toxinsare those that are nonlethal while maintaining T-cell mitogenicitycomparable to wild type SPE-A toxin. Preferred mutants are those thathave a change at residue 157 lysine of wild type SPE-A toxins such asK157E.

A pharmaceutical composition is formed by combining a mutant SPE-A toxinwith a physiologically acceptable carrier such as physiological saline,buffered saline solutions at neutral pH such as phosphate bufferedsaline. The mutant SPE-A toxin is combined in an amount effective tostimulate T-cell proliferation comparable to wild type SPE-A toxin atthe same dose. An enhancement in T-cell responsiveness can be measuredusing standard 3H thymidine assays with rabbit lymphocytes as well as bymeasuring T-cell populations in vivo using fluorescence activated T-cellsorters or an assay such as an ELISPOT. An effective amount can also bean amount effective to ameliorate or decrease the growth of cancercells. This can be determined by measuring the effect of the mutantSPE-A toxin on growth of cancer cells in vivo or by measuring thestimulation of cancer-specific T-cells. The range of effective amountsare 100 ng to 100 mg per kg of body weight, more preferably 1 μg to 1mg/kg body weight. About 10-6 μg of wild type SPE-A toxin can stimulateenhanced T cell responsiveness. For example, these mutant SPE-A toxinscould be used either alone or in conjunction with interleukin orinterferon therapy.

The invention also includes fragments of SPE-A toxins and fragments ofmutant SPE-A toxins. For vaccine compositions, fragments are preferablylarge enough to stimulate a protective immune response. A minimum sizefor a B cell epitope is about 4-7 amino acids and for a T cell epitopeabout 8-12 amino acids. The total size of wild type SPE-A is about 251amino acids including the leader sequence. Fragments are peptides thatare about 4 to 250 amino acids, more preferably about 10-50 amino acids.

Fragments can be a single peptide or include peptides from differentlocations joined together. Preferably, fragments include one or more ofthe domains as identified in FIG. 1 and as described previously. It isalso preferred that the fragments from mutant SPE-A toxins have at leastone change in amino acid sequence and more preferably 1-6 changes inamino acid sequence when compared to a protein substantiallycorresponding to a wild type SPE-A toxin.

Preferably, fragments are substantially nonlethal systemically.Fragments are screened and selected to have little or no toxicity inrabbits using the miniosmotic pump model at the same or greater dosagethan a protein having wild type SPE-A toxin activity as describedpreviously. It is also preferred that the fragment is nontoxic in humanswhen given a dose comparable to that of a wild type SPE-A toxin.

For vaccine compositions, it is preferred that the fragments includeresidues from the central α helix and/or the N-terminal a helix. It isespecially preferred that the fragment include a change at amino acidresidues equivalent to residue 20 in wild type SPE-A toxin such as N20Dor a change at an amino acid residue equivalent to residue 98 cysteinein a wild type SPE-A toxin.

For vaccine compositions, it is preferable that a fragment stimulate aneutralizing antibody response to a protein having wild type SPE-A toxinactivity. A fragment can be screened and selected for immunoreactivitywith polyclonal neutralizing antibodies to a wild type SPE-A toxin. Thefragments can also be used to immunize animals and the antibodies formedtested for neutralization of wild type SPE-A toxin.

For vaccine compositions, especially preferred fragments are furtherselected and screened to be nonbiologically active. By nonbiologicallyactive, it is meant that the fragment is nonlethal systemically, induceslittle or no enhancement of endotoxin shock, and induces little or no Tcell stimulation. Optionally, the fragment can be screened and selectedto have a decrease in capillary leak effect on porcine endothelialcells.

The fragments screened and selected for vaccine compositions can becombined into vaccine formulations and utilized as described previously.Optionally, fragments can be attached to carrier molecules such asbovine serum albumin, human serum albumin, keyhole limpet hemocyanin,tetanus toxoid and the like.

For pharmaceutical compositions, it is preferred that the fragmentsinclude amino acid residues in the N-terminal Domain B β strands 1-5alone or in combination with the central a helix. It is especiallypreferred if the fragments include a change at an amino acid residueequivalent to the lysine at amino acid 157 of a wild type SPE-A toxinsuch as K157E.

For pharmaceutical compositions, it is preferred that the fragments arescreened and selected for nonlethality systemically, and optionally forlittle or no enhancement of endotoxin shock as described previously. Itis preferred that the fragments retain T cell mitogenicity similar tothe wild type SPE-A toxin. Fragments of a mutant toxin SPE-A can formpharmaceutical compositions as described previously.

Fragments of mutant SPE-A toxin can be prepared using PCR, restrictionenzyme digestion and/or ligation, in vitro mutagenesis and chemicalsynthesis. For smaller fragments chemical synthesis may be desirable.

The fragments of mutant SPE-A toxins can be utilized in the samecompositions and methods as described for mutant SPE-A toxins.

B. Methods for Using Mutant SPE-A toxins, Vaccines Compositions orPharmaceutical compositions.

The mutant SPE-A toxins and/or fragments thereof are useful in methodsfor protecting animals against the effects of wild type SPE-A toxins,ameliorating or treating animals with STSS, inducing enhanced T-cellproliferation and responsiveness, and treating or ameliorating thesymptoms of cancer.

A method for protecting animals against at least one biological activityof wild type SPE-A toxin involves the step of administering a vaccinecomposition to an animal to establish a protective immune responseagainst at least one biological activity of SPE-A toxin. It is preferredthat the protective immune response is neutralizing and protects againstlethality or symptoms of STSS. The vaccine composition preferablyincludes a mutant SPE-A toxin or fragment thereof that has at least oneamino acid change, that immunoreacts with polyclonal neutralizingantibodies to wild type SPE-A, and is nonlethal. The especiallypreferred mutant has a change at amino acid residue 20 asparagine suchas the mutant N20D, or N20D/K157E or N20D/C98S.

The vaccine composition can be administered to an animal in a variety ofways including subcutaneously, intramuscularly, intravenously,intradermally, orally, intranasally, ocularly, intraperitoneally and thelike. The preferred route of administration is intramuscularly.

The vaccine compositions can be administered to a variety of animalsincluding rabbits, rodents, horses and humans. The preferred animal is ahuman.

The vaccine composition can be administered in a single or multipledoses until protective immunity against at least one of the biologicalactivities of wild type SPE-A is established. Protective immunity can bedetected by measuring the presence of neutralizing antibodies to thewild type SPE-A using standard methods. An effective amount isadministered to establish protective immunity without causingsubstantial toxicity.

A mutant SPE-A toxin or fragment thereof is also useful to generateneutralizing antibodies that immunoreact with the mutant SPE-A toxin andthe wild type SPE-A toxin. These antibodies could be used as a passiveimmune serum to treat or ameliorate the symptoms in those patients thathave the symptoms of STSS. A vaccine composition as described abovecould be administered to an animal such as a horse or a human until aneutralizing antibody response to wild type SPE-A is generated. Theseneutralizing antibodies can then be harvested, purified, and utilized totreat patients exhibiting symptoms of STSS. Neutralizing antibodies towild type SPE-A toxin can also be formed using wild type SPE-A. However,wild type SPE-A must be administered at a dose much lower than thatwhich induces toxicity such as 1/50 to 1/100 of the LD50 of wild typeSPE-A in rabbits.

The neutralizing antibodies are administered to patients exhibitingsymptoms of STSS such as fever, hypotension, group A streptococcalinfection, myositis, fascitis, and liver damage in an amount effectiveto neutralize the effect of SPE-A toxin. The neutralizing antibodies canbe administered intravenously, intramuscularly, intradermally,subcutaneously, and the like. The preferred route is intravenously orfor localized infection, topically at the site of tissue damage withdebridement. It is also preferred that the neutralizing antibody beadministered in conjunction with antibiotic therapy. The neutralizingantibody can be administered until a decrease in shock or tissue damageis obtained in a single or multiple dose. The preferred amount ofneutralizing antibodies typically administered is about 1 mg to 1000mg/kg, more preferably about 50-200 mg/kg of body weight.

The mutant SPE-A toxins and/or fragments thereof are also useful inpharmaceutical compositions for stimulation of T-cell proliferation,especially in the treatment of cancer. It is especially preferred thatthese pharmaceutical compositions be used in the place of or inconjunction with current therapies for cancer using interleukins,interferons or tumor necrosis factors. The mutant SPE-A toxins are alsouseful in treating T cell lymphomas, and ovarian and uterine cancer.While not meant to limit the invention, it is believed that mutant SPE-Atoxins can be selectively toxic for T lymphoma cells.

The pharmaceutical compositions include a mutant SPE-A toxin and/orfragment thereof that are nonlethal, while maintaining T cellmitogenicity. The preferred mutant SPE-A toxin is one that has a changeat amino acid residue 157 lysine such as K157E.

The pharmaceutical composition is administered to a patient havingcancer by intravenous, intramuscular, intradermal, orally,intraperitoneally, and subcutaneous routes, and the like. The preferredroute is intravenous. The pharmaceutical composition can be administeredin a single dose or multiple doses. The pharmaceutical composition isadministered in an amount that is effective to stimulate enhanced T-cellproliferative response and/or to decrease the growth of the cancerwithout substantial toxicity. The preferred amount ranges from 100 ng to100 mg/kg, more preferably 1 μg to 1 mg/kg. It is especially preferredthat the mutant SPE-A pharmaceutical compositions are administered inconjunction with or in place of therapies using interferons,interleukins, or tumor necrosis factors. C. DNA Expression CassettesEncoding Mutant SPE-A Toxins and Methods of Preparation of Such DNAExpression Cassettes The invention also includes DNA sequences andexpression cassettes useful in expression of mutant SPE-A toxins and/orfragments thereof. An expression cassette includes a DNA sequenceencoding a mutant SPE-A toxin and/or fragment thereof with at least oneamino acid change and at least one change in biological functioncompared to a protein substantially corresponding to a wild type SPE-Atoxin operably linked to a promoter functional in a host cell.Expression cassettes are incorporated into transformation vectors andmutant SPE-A toxins are produced in transformed cells. The mutant toxinscan then be purified from host cells or host cell supernatants.Transformed host cells are also useful as vaccine compositions.

Mutant SPE-A toxins or fragments thereof can also be formed by screeningand selecting for spontaneous mutants in a similar manner as describedfor site specific or random mutagenesis. Mutant SPE-A toxins can begenerated using in vitro mutagenesis or semisynthetically from fragmentsproduced by any procedure. Finally, mutant SPE-A toxins can be generatedusing chemical synthesis.

A method of producing the mutant SPE-C toxins or fragments thereof whichincludes transforming or transfecting a host cell with a vectorincluding such an expression cassette and culturing the host cell underconditions which permit expression of such mutant SPE-C toxins orfragments by the host cell.

DNA Sequences Encoding Mutant SPE-A Toxins

A mutant DNA sequence encoding a mutant SPE-A toxin that has at leastone change in amino acid sequence can be formed by a variety of methodsdepending on the type of change selected. A DNA sequence encoding aprotein substantially corresponding to wild type SPE-A toxin functionsas template DNA used to generate DNA sequences encoding mutant SPE-Atoxins. A DNA sequence encoding wild type SPE-A toxin is shown in FIG. 3and has been deposited in a microorganism with ATTC Accession number69830.

To make a specific change or changes at a specific location or locationsit is preferred that PCR is utilized according to method of Perrin etal., cited supra. To target a change to a particular location, internalprimers including the altered nucleotides coding for the amino acidchange are included in a mixture also including a 5′ and 3′ flankingprimers. A 5′ flanking primer is homologous to or hybridizes to a DNAregion upstream of the translation start site of the coding sequence forwild type SPE-A. Preferably, the 5′ flanking region is upstream of thespeA promoter and regulatory region. For example, a 5′ flanking primercan be homologous to or hybridize to a region about 760 bases upstreamof the translation start site as shown in FIG. 2. An example of a 5′flanking primer which includes the SPE-A promoter in upstream regulatoryregion has a sequence of: 5′ GGT GGA TTC TTG AAA CAG GTG-3′ (SEQ IDNO:1)         BamH1

A downstream flanking primer is homologous to or hybridizes to a regionof DNA downstream of the stop codon of the coding sequence for wild typeSPE-A. It is preferred that the downstream flanking primer provides fortranscriptional and translational termination signals. For example, a 3′flanking primer can hybridize or be homologous to a region 200 basepairs downstream of the stop codon for the coding sequence of SPE-A. Anexample of a 3′ flanking primer has a sequence: (SEQ ID NO:2) 5′ CCC CCCGTC GAC GAT AAA ATA GTT GCT AAG CTA             SalI CAA GCT-3′The upstream and downstream flanking primers are present in every PCRreaction to ensure that the resulting PCR product includes the speApromoter and upstream regulatory region and transcriptional andtranslation termination signals. Other upstream and downstream primerscan readily be constructed by one of skill in the art. While preferred,it is not absolutely necessary that the native speA promoter andupstream regulatory region be included in the PCR product.

Each mutation at a particular site is generated using an internal primerincluding a DNA sequence coding for a change at a particular residue.For example, amino acid substitutions at a specific site can begenerated using the following internal primers: Mutant Internal PrimerN2OD 5′ AAA AAC CTT CAA GAT ATA TAT TTT CTT-3′ (SEQ ID NO:3) C87S5′-TCC-ACA-TAA-ATA GCT GAG ATG GTA ATA- (SEQ ID NO:4) TCC-3′ C90S 5′-CTCTGT TAT TTA TCT GAA AAT GCA GAA-3′ (SEQ ID NO:5) C98S 5′ CCC TCC GTA GATCGA TGC ACT CCT TTC (SEQ ID NO:6) TGC-3′ K157E 5′-CTT ACA GAT AAT GAGCAA CTA TAT ACT-3′ (SEQ ID NO:7) 5195A 5′-CCA GGA TTT ACT CAA GCT AAATAT CTT (SEQ ID NO:8) ATG-3′ K16N 5′- CAA CTT CAC AGA TCT AGT TTA GTTAAC (SEQ ID NO:9) AAC CTT-3′(forward primer) and 5′- T TTG AAG GTT GTTAAC TAA ACT AGA (SEQ ID NO:10) TCT GTG AAG TTG-3′ (backward primer)The underlined nucleotides indicate changes in the nucleotide sequencefrom a wild type speA gene as shown in FIG. 3.

Internal primers can be designed to generate a change at a specificlocation utilizing a DNA sequence encoding wild type SPE-A toxins suchas shown in FIG. 3. Primers can be designed to encode a specific aminoacid substitution at a specific location such as shown above. Primerscan be designed to result in random substitution at a particular site asdescribed by Rennell et al., J. Mol. Biol. 22:67 (1991). Primers can bedesigned that result in a deletion of an amino acid at a particularsite. Primers can also be designed to add coding sequence for anadditional amino acid at a particular location.

Primers are preferably about 15 to 50 nucleotides long, more preferably15 to 30 nucleotides long. Primers are preferably prepared by automatedsynthesis. The 5′ and 3′ flanking primers preferably hybridize to theflanking DNA sequences encoding the coding sequence for the wild typeSPE-A toxin. These flanking primers preferably include about 10nucleotides that are 100% homologous or complementary to the flankingDNA sequences. Internal primers are not 100% complementary to DNAsequence coding for the amino acids at location because they encode achange at that location. An internal primer can have about 1 to 4mismatches from the wild type SPE-A sequence in a primer about 15 to 30nucleotides long. Both flanking primers and internal primers can alsoinclude additional nucleotides that encode for restriction sites andclamp sites, preferably near the end of the primer. Hybridizationconditions can be modified to take into account the number of mismatchespresent in the primer in accord with known principles as described bySambrook et al. Molecular Cloning-A laboratory manual, Cold SpringHarbor Laboratory Press, (1989).

More than one internal primer can be utilized if changes at more thanone site are desired. For example, to generate a mutant having a changeat amino acid 20 asparagine and a change at amino acid 157 lysineinternal primers as shown above can be utilized in two separatereactions as described in Example 5. A PCR method for generatingsite-specific changes at more than one location is described in Aiyar etal. cited supra. Another method is described in Example 5.

In one method, a DNA sequence encoding a mutant SPE-A toxin with onechange at a particular site is generated and is then used as thetemplate to generate a mutant DNA sequence with a change at a secondsite. In the first round of PCR, a first internal primer is used togenerate the mutant DNA sequence with the first change. The mutant DNAsequence with the first change is then used as the template DNA and asecond internal primer coding for a change at a different site is usedto form a DNA sequence encoding a mutant toxin with changes in aminoacid sequences at two locations. PCR methods can be utilized to generateDNA sequences with encoding amino acid sequences with about 2 to 6changes.

The preferred PCR method is as described by Perrin et al. cited supra.Briefly, the PCR reaction conditions are: PCR is performed in a 100 ulreaction mixture containing 10 mM Tris-HCl (pH=8.3), 50 mM KCl, 1.5 mMMgCl2, 200 uM each dNTP, 2 ng template plasmid DNA, 100 pmoles flankingprimer, 5 pmoles internal primer, and 2.5 units of Ampli Taq DNApolymerase (Perkin Elmer Cetus). In the second amplification step, thecomposition of the reaction mix is as above except for equal molarity (5pmoles each) of flanking primer and megaprimer and 1 ug template. PCR isconducted for 30 cycles of denaturation at 94° C.×1 minute, annealing at37° C. or 44° C.×2 minutes and elongation at 72° C. for 3 minutes.

The PCR products are isolated and then cloned into a shuttle vector(such as pMIN 164 as constructed by the method of Murray et al, J.Immunology 152:87 (1994) and available from Dr. Schlievert, Universityof Minnesota, Mpls, Minn.). This vector is a chimera of E. coli plasmidpBR328 which carries ampicillin resistance and the staphylococcalplasmid pE194 which confers erythromycin resistance. The ligated plasmidmixtures are screened in E. coli for toxin production using polylconalneutralizing antibodies to wild type SPE-A from Toxin Technologies, BocaRaton, Fla. or from Dr. Schlievert. The mutant SPE-A toxins aresequenced by the method of Hsiao et al., Nucleic Acid Res. 19:2787(1991) to confirm the presence of the desired mutation and absence ofother mutations.

Specific DNA sequences generated in this manner include a DNA sequencethat encodes mutant N20D and has the same coding sequence as shown inFIG. 3 except that an adenine at position 939 is changed to a guanineresidue. A DNA sequence that encodes mutant C87S has the same codingsequence of FIG. 3 except that thymine at position 1,152 is changed to aadenine and thymine at position 1,154 is changed to cytosine. A DNAsequence that encodes mutant SPE-A toxin C98S has the same codingsequence as FIG. 3 except that guanine at position 1,185 is changed tocytosine and thymine at position 1,186 is changed to guanine. A DNAsequence that encodes mutant SPE-A toxin C90S includes a sequence thathas the same coding sequence as FIG. 3 except that guanine at position1,161 is changed to a cytosine. A DNA sequence that encodes mutant SPE-Atoxin K157E includes a sequence that is the same as the coding sequenceshown in FIG. 3 but is changed at position 1,351 from adenine toguanine. A DNA sequence that encodes a mutant SPE-A toxin S195A includesa DNA sequence that has the same coding sequence as shown in FIG. 3except that thymine at position 1,464 is a guanine. A DNA sequence thatencodes a mutant K16N SPE-A toxin includes a sequence that is the sameas that shown in FIG. 3 except that adenine at position 941 is changedto cytosine.

It will be understood by those of skill in the art that due to thedegeneracy of the genetic code a number of DNA sequences can encode thesame changes in amino acids. The invention includes DNA sequences havingdifferent nucleotide sequences but that code for the same change inamino acid sequence.

For random mutagenesis at a particular site a series of primers aredesigned that result in substitution of each of the other 19 amino acidsor a non-naturally occurring amino acid or analog at a particular site.PCR is conducted in a similar manner as described above or by the methoddescribed by Rennell et al., cited supra. PCR products are subcloned andthen toxin production can be monitored by immunoreactivity withpolylconal neutralizing antibodies to wild type SPE-A. The presence of achange in amino acid sequence can be verified by sequencing of the DNAsequence encoding the mutant SPE-A toxin. Preferably, mutant toxins arescreened and selected for nonlethality.

Other methods of mutagenesis can also be employed to generate randommutations in the DNA sequence encoding the wild type SPE-A toxin. Randommutations or random mutagenesis as used in this context means mutationsare not at a selected site and/or are not a selected change. A bacterialhost cell including a DNA sequence encoding the wild type SPE-A toxin,preferably on pMIN 164, can be mutagenized using other standard methodssuch as chemical mutagenesis, and UV irradiation. Mutants generated inthis manner can be screened for toxin production using polyclonalneutralizing antibodies to wild type SPE-A. However, further screeningis necessary to identify mutant toxins that have at least one change ina biological activity, preferably that are nonlethal. Spontaneouslyarising mutants can also be screened for at least one change in abiological activity from wild type SPE-A.

Random mutagenesis can also be conducted using in vitro mutagenesis asdescribed by Anthony-Cahill et al., Trends Biochem. Sci. 14: 400 (1989).

In addition, mutant SPE-A toxins can be formed using chemical synthesis.A method of synthesizing a protein chemically is described in Wallace,FASEB J. 7:505 (1993). Parts of the protein can be synthesized and thenjoined together using enzymes or direct chemical condensation. Usingchemical synthesis would be especially useful to allow one of skill inthe art to insert non-naturally occurring amino acids at desiredlocations. In addition, chemical synthesis would be especially usefulfor making fragments of mutant SPE-A toxins.

Any of the methods described herein would be useful to form fragments ofmutant SPE-A toxins. In addition, fragments could be readily generatedusing restriction enzyme digestion and/or ligation. The preferred methodfor generating fragments is through direct chemical synthesis forfragment of 20 amino acids or less or through genetic cloning for largerfragments.

DNA sequences encoding mutant toxins, whether site-specific or random,can be further screened for other changes in biological activity fromwild type SPE-A toxin. The methods for screening for a change in atleast one biological activity are described previously. Once selectedDNA sequences encoding mutant SPE-A toxins are selected for at least onechange in biological activity, they are utilized to form an expressioncassette.

Formation of an expression cassette involves combining the DNA sequencescoding for mutant SPE-A toxin with a promoter that provides forexpression of a mutant SPE-A toxin in a host cell. For those mutantSPE-A toxins produced using PCR as described herein, the native speApromoter is present and provides for expression in a host cell.

Optionally, the DNA sequence can be combined with a different promoterto provide for expression in a particular type of host cell or toenhance the level of expression in a host cell. Preferably, the promoterprovides for a level of expression of the mutant SPE-A toxin so that itcan be detected with antibodies to SPE-A. Other promoters that can beutilized in prokaryotic cells include PLAC, PTAC, T7, and the like.

Once the DNA sequence encoding the mutant SPE-A toxin is combined with asuitable promoter to form an expression cassette, the expressioncassette is subcloned into a suitable transformation vector. Suitabletransformation vectors include at least one selectable marker gene andpreferably are shuttle vectors that can be amplified in E. coli and grampositive microorganisms. Examples of suitable shuttle vectors includepMIN 164, and pCE 104. Other types of vectors include viral vectors suchas the baculovirus vector, SV40, poxviruses such as vaccinia, adenovirusand cytomegalovirus. The preferred vector is a pMIN 164 vector, ashuttle vector that can be amplified in E. coli and S. aureus.

Once a transformation vector is formed carrying an expression cassettecoding for a mutant SPE-A toxin, it is introduced into a suitable hostcell that provides for expression of the mutant SPE-A toxin. Suitablehost cells are cells that provide for high level of expression of themutant toxin while minimizing the possibility of contamination withother undesirable molecules such as endotoxin and M-proteins. Suitablehost cells include mammalian cells, bacterial cells such as S. aureus,E. coli and Salmonella spp., yeast cells, and insect cells.

Transformation methods are known to those of skill in the art andinclude protoplast transformation, liposome mediated transformation,calcium phosphate precipitation and electroporation. The preferredmethod is protoplast transformation.

Preferred transformed cells carry an expression cassette encoding amutant SPE-A toxin with a change at amino acid 20 asparagine. Such atransformed cell has been deposited with the American Type CultureCollection in Rockville, Md. The characteristics of the depositedmicroorganism is that it is a S. aureus carrying pMIN 164 including aDNA sequence encoding mutant N20D operably linked to the native speApromoter and other regulatory regions. This microorganism was depositedin accordance with the Budapest treaty and given Accession number 69831.

Another microorganism has been deposited with the ATCC. Thismicroorganism is S. aureus carrying a DNA sequence encoding the wildtype SPE-A toxin operably linked to the native speA promoter andregulatory regions. This microorganism was deposited with the ATCC inaccord with the Budapest treaty and given Accession number 69830.

Transformed cells are useful to produce large amounts of mutant SPE-Atoxin that can be utilized in vaccine compositions. A transformedmicroorganism can be utilized in a live, attenuated, or heat killedvaccine. A transformed microorganism includes mutant toxin SPE-A inamounts sufficient to stimulate a protective immune response to wildtype SPE-A. Preferably, the mutant SPE-A toxin is secreted. Themicroorganism is preferably nonpathogenic to humans and includes amutant toxin with multiple amino acid changes to minimize reversion to atoxic form. The microorganism would be administered either as a live orheat killed vaccine in accordance with known principles. Preferredmicroorganisms for live vaccines are transformed cells such asSalmonella spp.

A viral vector including an expression cassette with a DNA sequenceencoding a mutant SPE-A toxin or fragment thereof operably linked to apromoter functional in a host cell can also be utilized in a vaccinecomposition as described herein. Preferably, the promoter is functionalin a mammalian cell. An example of a suitable viral vector includes poxviruses such as vaccinia virus, adenoviruses, cytomegaloviruses and thelike. Vaccinia virus vectors could be utilized to immunize humansagainst at least one biological activity of a wild type SPE-A toxin.

The invention also includes a vaccine composition comprising an nucleicacid sequence encoding a mutant SPE-A toxin or fragment thereof operablylinked to a promoter functional in a host cell. The promoter ispreferably functional in a mammalian host cell. The nucleic acidsequence can be DNA or RNA. The vaccine composition is delivered to ahost cell or individual for expression of the mutant SPE A toxin orfragment thereof within the individuals own cells. Expression of nucleicacid sequences of the mutant SPE A toxin or fragment thereof in theindividual provides for a protective immune response against the wildtype SPE A toxin. Optionally, the expression cassette can beincorporated into a vector. A nucleic acid molecule can be administeredeither directly or in a viral vector. The vaccine composition can alsooptionally include a delivery agent that provides for delivery of thevaccine intracellularly such as liposomes and the like. The vaccinecomposition can also optionally include adjuvants or otherimmunomodulatory compounds, and additional compounds that enhance theuptake of nucleic acids into cells. The vaccine composition can beadministered by a variety of routes including parenteral routes such asintravenously, intraperitoneally, or by contact with mucosal surfaces.

Conditions for large scale growth and production of mutant SPE-A toxinare known to those of skill in the art. A method for purification ofmutant SPE-A toxins from microbial sources is as follows. S. aureuscarrying the mutant or the wild type speAs in pMIN164 are grown at 37°C. with aeration to stationary phase in dialyzable beef heart medium,containing 5 mg/ml of erythromycin. Cultures are precipitated with fourvolumes of ethanol and proteins resolubilized in pyrogen free water. Thecrude preparations are subjected to successive flat bed isoelectricfocusing separations in pH gradients of 3.5 to 10 and 4 to 6. Thefractions that are positive for toxin by antibody reactivity areextensively dialyzed against pyrogen free water, and an aliquot of eachis tested for purity by SDS polyacrylamide gel electrophoresis in 15%(weight/volume) gels. Polyclonal neutralizing antibodies to SPE-A areavailable from Toxin Technologies, Boca Raton, Fla. or Dr. Schlievert.Other methods of purification including column chromatography or HPLCcan be utilized.

This invention can be better understood by way of the following exampleswhich are representative of the preferred embodiments thereof, but whichare not to be construed as limiting the scope of the invention.

EXAMPLE 1 Cloning and Expression of SPE-A Wild Type

The gene encoding wild type SPE-A toxin (speA) was cloned from E. colias described in Johnson et al., Mol. Gen. Genet. 194:52-56 (1984).Briefly, the speA gene was identified by cloning of a HindIII digest ofPhage T12 DNA in pBR322 in E. Coli RR1. Transformants were selected byidentifying those positive for toxin production using polylconalneutralizing antisera to A toxin. A nucleotide sequence for A toxin isreported in Weeks et al, Inf. Imm. 52: 144 (1986).

A DNA sequence including the speA gene was subcloned and then expressedin S. aureus. The speA carried on a E. coli plasmid was digested withrestriction enzymes HindIII and SalI. The fragments were purified andligated into HindIII-SalI sites of pMIN 164 (available as describedpreviously). The vector pMIN 164 is a chimera of the staphylococcalplasmid pE194 (carrying erythromycin resistance) and the E. coli vectorpBR328 (carrying Amp and Tet resistance). Cloning of speA into theHindIII-SalI sites of this vector disrupts Tet resistance. The promoterpresent in this plasmid immediately upstream of the cloned gene is thenative speA promoter.

Expression of the speA gene was verified by detecting the toxin in adouble immunodiffusion assay with polyclonal neutralizing antibodies toSPE-A from Toxin prepared in the inventors laboratory.

EXAMPLE 2 Administration and Immunization of Rabbits with RecombinantlyProduced SPE-A (wt)

Administration of recombinantly produced SPE-A to animals induces STSS.Immunization of animals with recombinantly produced SPE-A reduces thedeath rate when animals are challenged with M3 or M1 streptococci andprotects animals against STSS.

Administration of SPE-A induces STSS in rabbits. A rabbit model for STSShas been established by administration of SPE-A in subcutaneouslyimplanted miniosmotic pumps. Lee et al., Infect Immun. 59:879 (1991).These pumps are designed to release a constant amount of toxin over a7-day period, thus providing continuous exposure to the toxin.Recombinantly produced SPE-A was administered to rabbits at a total doseof 200 μg/in 0.2 ml over a 7-day period. The results indicate thatanimals treated with SPE-A developed the criteria of STSS with nearlyall animals succumbing in the 7-day period (data not shown). Thesymptoms of STSS in rabbits include weight loss, diarrhea, mottled face,fever, red conjunctiva and mucosa, and clear brown urine. As expected,control non-toxin treated animals remained healthy. Two other majorobservations were made: 1) fluid replacement provided completeprotection to the animals as expected, and 2) none of the toxin treatedanimals developed necrotizing fascitis and myositis, indicating factorsother than, or in addition to, SPE-A are required for the soft tissuedamage.

Development of the clinical features of STSS correlates withadministration of SPE-A. Rabbits injected with SPE-A positivestreptococci developed STSS whereas those injected with SPE-A negativestreptococci did not show symptoms of STSS.

It is well known that SPE-A is a variable trait made by some group Astreptococci. The gene for SPE-A is encoded by bacteriophage T12, andwell-characterized streptococcal strains were established that differonly in whether or not the SPE-A phage, referred to as T12 phage, ispresent. Streptococcal strain T253 cured T12 is positive for productionof SPE-A, whereas T253 cured is SPE-A negative.

Rabbits were injected subcutaneously with SPE-A positive streptococciT253 cured T12 or SPE-A negative T253 cured into implanted Wiffle golfballs, as described by Scott et al., Infect Immunity 39:383 (1983). Theresults are shown in Table 1. The results show that animals injectedwith SPE-A positive streptococci developed the clinical features ofSTSS, and 6/8 succumbed. The two surviving animals developed antibodiesto SPE-A. In contrast, the toxin negative strain, T253 cured, inducedonly fever, and no deaths were observed, even at much higher bacterialcell concentrations. As in the previous animal model experiments, noevidence of soft tissue necrosis was observed. Furthermore, thestreptococci remained localized in the golf balls, suggesting thesestreptococcal strains were not highly invasive. TABLE 1 Induction ofSTSS by speA in a Wiffle ball Rabbit Model Average Highest TreatmentTemperature (° C.) Dead/Total None 39.1 0/4 T253 cured T12* 41.2 6/8^(I) T253 cured* 40.7 0/6 T253 cured+ 41.0 0/6*Approximately 1 × 108 cells+Approximately 1 × 1011 cells^(I)2 survivors developed antibodies to SPE-A

Immunization with recombinantly produced SPE-A decreased death rateswhen rabbits were challenged with M1 or M3 streptococci. Rabbits wereimmunized with cloned SPE-A derived from S. aureus to prevent thepossibility of immunizing the animals with contaminating streptococcalproducts, such as M protein. Control animals were not immunized againstSPE-A. The rabbits received 50 μg of recombinantly produced SPE-A inemulsified in Freund's incomplete adjuvant subcutaneously. After 9 days,rabbits were challenged subcutaneously with 25 ml of M3 (1.4×109 totalCFU) or M1 (4.2×109 total CFU) streptococci grown in dialyzed beef heartmedium. The M1 and M3 streptococcal isolates are clinical isolates. TheM1 isolate is designated MNST and the M3 isolate is designated MNBY.These isolates are available from Dr. Schlievert, University ofMinnesota, Mpls. MN.

The data presented in Table 2 show the striking results of theseexperiments. TABLE 2 Protection of Rabbits from STSS with necrotizingfascitis and myositis, induced by M3 or M1 streptococci, by priorimmunization against SPE-A Number of Immunizing Challenge NumberAlive^(I) Animals Agent* Agent⁺ Total 20 — M3  4/20 P << 0.001′ 20 SPE-AM3 16/19 17 — M1  9/17 P < 0.04′ 15 SPE-A M1 13/15*Animals were immunized against cloned SPE-A prepared from S. aureus;ELISA titers against SPE-A were greater than 10,000.⁺Animals were challenged subcutaneously with 1.4 × 109 CFU M3 or 4.2 ×109 CFU M1 streptococci in a dialyzable beef heart medium.^(I)According to the guidelines of the University of Minnesota AnimalCare Committee, the experiment which used M3 streptococci was terminatedafter 24 h, and the experiment that used M1 streptococci was terminatedafter 48 h.′P values determined by Fisher's Exact Probability Test.

As indicated 16 of 19 SPE-A immunized rabbits survived challenge with M3streptococci, whereas only 4 of 20 nonimmune animals survived. Thesurviving 30 immune animals showed clear evidence of contained softabscess formation, upon which examination of the fluid, was filled withPMNs. Similar results were obtained in studies of M1 streptococci,except the M1 organisms were not as virulent as the M3 organisms (Table2). Higher numbers of M1 streptococci were used, and a reduced deathrate in the rabbits was seen, even in nonimmune control animals. Thismay reflect the approximately 50-fold lower SPE-A production by M1strains compared to M3 strains.

In contrast, none of the nonimmune animals showed abscess formation, andexamination of fluid from 2/2 animals revealed no PMN infiltrate. Theseresults show that one major difference between the SPE-A immune versusnonimmune animals appears to be whether or not an inflammatory responsecould be mounted. Prior work showed that SPE-A, as well as otherpyrogenic toxin superantigens, induce macrophages to produce high levelsof TNF-α. TNF-α greatly reduces PMN chemotaxis, apparently through downregulation of chemotactic receptors. Therefore, it is believed that theresults show that antibodies in the SPE-A immunized animals(titers >10,000 by ELISA) block the release of TNF-α from macrophages byneutralizing SPE-A, thus allowing the development of a protectiveinflammatory response. In the nonimmune animals SPE-A could cause asignificant release of TNF-α which in turn prevents development of aprotective chemotactic response.

It is important to note that all of the animals that died except oneshowed extensive soft tissue damage as evidenced by their entire sidesturning purple-black and in many cases sloughing. One animal in theimmunized group died after immunization. The lack of detectableinflammation in the tissue of these animals suggest that streptococcalfactors and not components of a host immune response causes necrotizingfascitis and myositis. Other extracellular factors may also contributeto the soft tissue damage, such as SPE B and streptolysins O and S.

All of the above data make a strong case for the causative role ofpyrogenic toxin superantigens, and particularly SPE-A, when present, inthe development of STSS.

EXAMPLE 3 Site Directed Mutagenesis of a DNA Sequence Encoding SPE-A

Locations in the SPE-A molecule important for biological activity wereidentified using site directed mutagenesis. Single amino acid changeswere introduced into various regions of the molecule as described below.

The model of the three dimensional structure of SPE-A is shown inFIG. 1. This model structure was constructed by Homology using anInsight/Homology program from BioSym Corp., San Diego, Calif. Thismolecule has several domains identified as: Domain Corresponding AminoAcids Helix 2 11-15 N terminal {acute over (α)}-helix, helix 3 18-26Domain B - β strands strand 1 30-36 strand 2 44-52 strand 3 55-62 strand4 75-83 strand 5  95-106 Central α-helix, helix 5 142-158 Domain A - βstrands strand 6 117-126 strand 7 129-135 strand 8 169-175 strand 9180-186 strand 10 213-220 Helix 4 64-72 Helix 6 193-202

Amino acids were selected in each of the domains and to alter thecysteine residues in the molecule. The especially preferred regions arethe N terminal α-helix (18-26); the central α-helix (142 to 158); DomainA β strands and Domain B β strands.

Target residues for mutagenesis were chosen among the conserved aminoacids throughout the pyrogenic toxin family by comparing primary aminoacid sequence and/or 3-D conformational similarities or homologies usingcomputer programs as described previously. The changes made to each ofthe amino acids were selected to change the characteristics of the aminoacid side chain of residue at the particular site. For example, at threeof the residues (87, 90 and 98) serine was substituted for cysteine soas to alter the sulphydryl groups in the molecule. At three other aminoacid residues changes were made in the charge present at that site. Forexample, a lysine was changed to a glutamic (157) acid, lysine waschanged to asparagine (16) and asparagine was changed to aspartic acid(20).

Other amino acids may affect the interaction of the toxins with MHCClass II molecules. In another molecule, the TSST-1 N terminal β barrelstrands were important for contacts with a and β chains of MHC class IImolecules. Therefore, changes in the Domain A and Domain B β strands maybe important for controlling the interaction of these molecules with MHCClass II molecules. In addition, changes in the residues can be preparedusing random mutagenesis and substitution of each of the other 19 aminoacids at a particular location, and then selecting those mutants showingan alteration in biological activity such as lethality.

The mutant SPE-A molecules were prepared using site directed mutagenesisusing polymerase chain reaction (PCR) in which the template DNA was thecloned SPE-A gene from phage T12. These primers were utilized for eachmutation generated. Generation of each mutant involved using threeprimers as follows: an upstream 5′ flanking primer, an internal primerincluding the change in DNA sequence coding for a change in an aminoacid and a downstream flanking primer. The upstream flanking primer wasincluded in every PCR reaction and is homologous to a DNA region about760 bases upstream of the translational start site and has a sequence:(SEQ ID NO:11) 5′ GGT GGA TCC TTG AAA CAG GTG CA-3′        BamH1

The resulting PCR product includes the speA promoter and possibleupstream regulatory region. The downstream flanking primer iscomplementary to a region of DNA about 270 bases downstream of the stopcodon and has a sequence: (SEQ ID NO:2) 5′ -CCC CCC GTC GAC GAT AAA ATAGTT GCT AAG CTA                Sal I CAA GCT-3′The downstream flanking primer is present in every PCR reaction andbecause of the location of the primer the PCR product contains aputative transcription termination sequence.

Each mutation is generated using an internal primer including a DNAsequence coding for a change at a particular amino acid residue. Theinternal primers used to generate each mutant are as follows: MutantInternal Primer N2OD 5′ AAA AAC CTT CAA GAT ATA TAT TTT CTT-3′ (SEQ IDNO:3) C87S 5′-TCC-ACA-TAA-ATA GCT GAG ATG GTA ATA- (SEQ ID NO:4) TCC-3′C90S 5′-CTC TGT TAT TTA TCT GAA AAT GCA GAA-3′ (SEQ ID NO:5) C98S 5′ CCCTCC GTA GAT CGA TGC ACT CCT TTC (SEQ ID NO:6) TGC-3′ K157E 5′-CTT ACAGAT AAT GAG CAA CTA TAT ACT-3′ (SEQ ID NO:7) 5195A 5′-CCA GGA TTT ACTCAA GCT AAA TAT CTT (SEQ ID NO:8) ATG-3′ K16N 5′- CAA CTT CAC AGA TCTAGT TTA GTT AAC (SEQ ID NO:9) AAC CTT-3′(forward primer) and 5′- T TTGAAG GTT GTT AAC TAA ACT AGA (SEQ ID NO:10) TCT GTG AAG TTG-3′ (backwardprimer)The underlined residues indicate changes in coding sequence made fromDNA sequence coding will type SPE-A.

PCR was conducted as follows: Briefly, a downstream flanking primer anda forward primer spanning the site of mutation and containing thenucleotide substitutions necessary to generate an amino acid change weremixed in unequal molarity in a standard PCR reaction. The DNA productobtained was prevalent in the strand containing the mutation. Thisproduct, or megaprimer, that can be several hundred bases long, wasisolated by electrophoresis in 1% agarose gel and eluted by the use ofthe Geneclean kit, as recommended by the manufacture (Bio 101, La Jolla,Calif.).

Briefly, the PCR reaction conditions are: PCR is performed in a 100 ulreaction mixture containing 10 mM Tris-HCl (pH=8.3), 50 mM KCl, 1.5 mMMgCl2, 200 uM each dNTP, 2 ng template plasmid DNA, 100 pmoles flankingprimer, 5 pmoles internal primer, and 2.5 units of Ampli Taq DNApolymerase (Perkin Elmer Cetus). In the second amplification step, thecomposition of the reaction mix is as above except for equal molarity (5pmoles each) of flanking primer and megaprimer and 1 ug template. PCR isconducted for 30 cycles of denaturation at 94° C.×1 minute, annealing at37° C. or 44° C.×2 minutes and elongation at 72° C. for 3 minutes.Hybridization conditions can be varied in accord with known principlesdepending on the primer size, mismatches, and GC content.

A plasmid containing the speA cloned gene and flanking sequences wasused as a template. In the second step, the megaprimer and an upstreamflanking primer were combined in the reaction mixture in equal molarityto generate the full length mutant speA.

The mutant speAs were digested with appropriate restriction enzymes andcloned into the shuttle vector pMIN 164. This vector is a chimera of theE. coli plasmid pBR328, which carries an ampicillin resistance gene, andthe staphylococcal plasmid pE194, which confers erythromycin resistance.The ligated plasmid mixtures were transformed, selected for, andscreened in E. coli. Clones positive for toxin production, as judged bydouble immunodiffusion assays, were sequenced by the method of Hsiaocited supra to confirm the presence of the desired mutation and theabsence of other mutations. Plasmids were then transformed in S. aureusstrain RN 4220 (available from Richard Novick, Skirball Institute, NewYork, N.Y.) for expression and production of mutant toxins.

S. aureus carrying the mutant or the wild type speAs in pMIN164 weregrown at 37° C. with aeration to stationary phase in dialyzable beefheart medium, containing 5 μg/ml of erythromycin. Cultures wereprecipitated with four volumes of ethanol and proteins resolubilized inpyrogen free water. The crude preparations were subjected to successiveflat bed isoelectric focusing separations in pH gradients of 3.5 to 10and 4 to 6. The fractions that were positive for toxin by antibodyreactivity were extensively dialyzed against pyrogen free water, and analiquot of each was tested for purity by SDS polyacrylamide gelelectrophoresis in 15% (weight/volume) gels (data not shown). Allmutants prepared were as resistant as the native toxin to treatment for60 minutes with trypsin (2 μg/μg SPE-A), and this together with theconserved reactivity to polyclonal antibodies raised against nativeSPE-A indicates that the mutations introduced do not cause grossstructural changes of the toxin. Using these methods, 7 mutants havingsingle amino acid substitutions in the amino acid sequence of SPE-A weregenerated.

EXAMPLE 4 Biological Activity Profile of Mutant SPE-A

Biological activities of the mutant toxins were evaluated and comparedto those of the wild type SPE-A. The mutant toxins were tested for theability to stimulate proliferation of T lymphocytes (superantigenicity),to enhance host susceptibility to endotoxin shock and for development oftoxic shock syndrome and lethality.

The ability to stimulate proliferation of T lymphocytes was measured as[3H] thymidine incorporation into cellular DNA of rabbit splenocytes. Astandard 4-day mitogenicity assay was performed in 96 well microtiterplates. Each well contained 2×105 rabbit splenocytes resuspended in 200μl RPMI 1640 (Gibco, Grand Island, N.Y.) supplemented with 25 mM HEPES,2.0 mM L-glutamine, 100 U penicillin, 100 μg/ml streptomycin and 2% heatinactivated FCS. 20 μl samples of exotoxins were added in quadruplicateamounts in final amounts: 1 μg to 10-5 μg/well. The background cellularproliferation was determined in quadruplicate wells by adding 20 μl RPMIto the splenocytes. After 3 days of incubation in a humidified chamberat 37° C. and 7% CO2, 1.0 μCi (20 μl volume of 5-[methyl-3H]-thymidine(46 Ci/mmole, Amersham, Arlington Heights, Ill.) was added to each welland incubated for 18 hours. Cellular DNA was collected on glass fiberfilters and the [methyl-3H] thymidine incorporation was quantified byliquid scintillation counting. Three separate assays using threedifferent rabbit donors were performed. Exoprotein concentrations weretested in quadruplicate in each of three assays. Results are presentedas CPM.

The ability to enhance host susceptibility to endotoxin shock was testedin American Dutch Belted rabbits. Animals weighing between 1 and 2 kgwere injected in the marginal ear vein with 5 μg/kg body weight of SPE-A(equal to 1/50 LD50) and challenged 4 hours later by IV injection of 1or 10 μg/kg body weight of endotoxin (about 1/100 LD50) from Salmonellatyphimurium. Control rabbits received injections with PBS. The animalswere monitored after 48 hours for death.

Lethality was also measured using miniosmotic pumps implantedsubcutaneously in American Dutch Belted rabbits and containing 200 μg oftoxin. Individual proteins (200 μg) were injected in 0.2 ml PBS intominiosmotic pumps (Alzet, AlzaCo, Palo Alto, Calif.). The pump isdesigned to deliver a constant amount of toxin over a 7-day period.Rabbits were monitored 3 times daily for signs of toxic shock syndromesuch as diarrhea, erythema of conjunctivae and ears, shock and death forup to 8 days.

The results of the T cell mitogenicity studies are shown in FIGS. 4, 5and 6. The results show that the mutant N20D had a five-fold decrease insuperantigenicity or T cell mitogenicity activity. Mutants C87S and C98Salso had a 4-fold decrease in mitogenicity for T cells. Thus, several ofthe mutations affected biological activity of superantigenicity or Tcell mitogenicity.

The results of enhancement of endotoxin shock and lethality are shown inTables 3, 4, and 5 shown below. TABLE 3 Mutants SPE-A-K16N andSPE-A-N20D assayed for ability to cause endotoxin enhancement orlethality when administered in subcutaneous miniosmotic pumps. Resultsare expressed as ratio of deaths over total rabbits tested Protein SPE-AK16N N20D Endotoxin enhancement 3/3 6/7 0/3 1 μg/kg endotoxin) Lethalityin miniosmotic pumps 3/4 ND 0/4

TABLE 4 Mutants SPE-A-C87S, SPE-A-C90S, and SPE-A-C98S tested forability to induce endotoxin enhancement or lethality when administeredin subcutaneous miniosmotic pumps. Results are expressed as ratio ofdeaths over total number of treated rabbits. Protein SPE-A C87S C98SC90S Endotoxin enhancement 2/3 1/3 0/3 ND  1 μg/kg body weight Endotoxinenhancement 2/3 3/3 1/3 ND 10 μg/kg body weight Lethality in miniosmoticpumps 3/4 ND ND 3/3

TABLE 5 Mutants SPE-A-K157E and SPE-A-S195A tested for ability to inducelethality when administered in subcutaneous miniosmotic pumps. Resultsare expressed as ratio of deaths over total number of treated rabbitsProtein SPE-A K157E S195A Lethality in miniosmotic pumps 6/8 0/4 3/3

The results show that animals treated with the mutant N20D did notdevelop STSS when tested using either model of STSS. The mutation inN20D is located in an organized α-helix bordering the deep groove on theback of the toxin (FIG. 1). This residue is important both insuperantigenicity and lethality functions of the molecule.

Mutations that eliminated sulphydryl groups and, therefore, thatinterfere with possible disulfide linkages, have varied effects on thebiological activities of SPE-A, depending on which cysteine residue wasmutated. The C90S mutant remained completely lethal (Table 4), and Tcell stimulatory activity was not significantly decreased (FIG. 5 a). Incontrast, C87S and C98S mutations reduced approximately four fold thetoxin's mitogenicity (FIG. 5 b). However, ability to cause endotoxinshock was affected differently by the two mutations, with C98S beingonly weakly toxic, but C87S being strongly toxic (Table 4). Anexplanation for these results is based upon the relative positions ofthe three cysteine residues in the primary sequence and in the3-dimensional structure (FIG. 1). The lack of the sulfhydryl group ofC98 may preclude formation of a putative disulfide bridge seen instaphylococcal enterotoxins, and therefore, the conformation of the loopwould be lost. This would have detrimental effects for the activity ifamino acids in this loop are responsible for contact with host cellularreceptors or have some other function in biological activity of themolecule. In the case of C87S mutation, the putative disulfide bondcould still be created between C90 and C98, preserving most of theconformation and, therefore, the activity.

Mutant K157E, located within the long central a-helix, retained completesuperantigeriicity (FIG. 6 b), but was nonlethal when administered inminiosmotic pumps to rabbits (Table 6).

Residue S195A, which is part of a-5 helix, may not be important for thebiological activities tested, since its mutation does not affectactivities tested thus far. This residue may not be exposed to theenvironment or may not contribute to binding.

These results show that lethality and superantigenicity can be affectedby mutations at several sites. Lethality can be affected by mutations inresidues in the N terminal α-helix (N20D) and in the central α-helix(K157E). Mitogenicity can be affected by mutations in the N terminalα-helix and changes to sulfhydryl groups.

These results also show that mitogenicity and lethality are separableactivities as mutants were generated that affect lethality withoutaffecting superantigenicity (K157E) and that affected mitogenicitywithout affecting lethality (C87S).

EXAMPLE 5 Preparation of Double or Triple Mutants of SPE-A Using PCR

There are a number of methods that can be used to generate double ortriple mutant SPE-A toxins or fragments thereof.

Mutant SPE-A toxins with two or more changes in amino acid sequenceswere prepared using PCR as described previously. In a first PCRreaction, an first internal primer coding for the first change at aselected site was combined with 5′ and 3′ flanking primers to form afirst PCR product. The first PCR product was a DNA sequence coding for amutant SPE-A toxin having one change in amino acid sequence. This firstPCR product then served as the template DNA to generate a second PCRproduct with two changes in amino acid sequence compared with a proteinhaving wild type SPE-A activity. The first PCR product was the templateDNA combined with a second internal primer coding for a change in aminoacid at a second site. The second internal primer was also combined withthe 5′ and 3′ flanking primers to form a second PCR product. The secondPCR product was a DNA sequence encoding a mutant SPE-A toxin withchanges at two sites in the amino acid sequence. This second PCR productwas then used as a template in a third reaction to form a product DNAsequence encoding a mutant SPE-A toxin with changes at three sites inthe amino acid sequence. This method was utilized to generate DNAsequences encoding mutant toxins having more than one change in theamino acid sequence.

An alternative method to prepare DNA sequences encoding more than onechange is to prepare fragments of DNA sequence encoding the change orchanges in amino acid sequence by automated synthesis. The fragments arethen subcloned into the wild type SPE-A coding sequence using severalunique restriction sites. Restriction sites are known to those of skillof the art and are readily determined from the DNA sequence of a wildtype SPE-A toxin. The cloning is done in a single step with a threefragment ligation method as described by Revi et al. Nucleic Acid Res.16: 1030 (1988).

Mutant D45N was obtained by the in vitro site directed mutagenesissystem Altered Sites II (Promega, Madison, Wis.). The 1.75 kb BamHI-SalI fragment of speA was subcloned in vector pAlter provided in themutagenesis kit (Promega). The mutagenic oligonucleotide was CTT TTA TCTCAC AAT TTA ATA TAT AAT G. The mutagenesis reactions were performed assuggested by the manufacturer.

Generation of Triple Mutants

Single amino acid mutants, such as D45N described immediately above,were used to produce double mutants and the triple mutant by subcloningfragments of speA carrying the desired new mutation into plasmids withsingle or double speA mutations. Table 10 describes the uniquerestriction sites used for the swapping of DNA segments and therecipient plasmid for each subcloning procedure. The mutants weresequenced in the region of the newly introduced mutation to confirm thesubcloning was successful. TABLE 10 List of multiple mutationsintroduced in SPE A and restriction fragments swapped to generatemultiple mutants from single mutants. Mutant Restriction Fragment DonorRecipient N20D/D45N/C98S Step 1 SalI-BstEII D45N N20D Step 2 BamHI-BfrIN20D/D45N C98S

EXAMPLE 6 Toxicity Studies related to Single and Double Mutants

Wild type SPE A, SPE A N20D, SPE A K157E, SPE A N20/C98S, and SPE AN20D/K157E were evaluated for superantigenicity based on their capacityto stimulate rabbit splenocyte proliferation (see FIGS. 7 and 8).

Double mutants SPE A (N20D/C98S, N20D/K157E) were prepared by PCRmutagenesis using the techniques described above. The mutant SPE A gene,speA N20D, served as template DNA for introduction of the secondmutation. The double mutant genes were sequenced as described above toinsure that only the indicated changes were present. Only the desiredchanges were present.

Rabbit spleen cells were cultured in the presence of SPE A and SPE Amutants in vitro for 3 days and then an additional day after addition of1 μCi/well of 3H thymidine. Incorporation of 3H thymidine intolymphocyte DNA was used as the measure of T cell proliferation. Asuperantigenicity index was calculated as average counts/min 3Hthymidine incorporation in stimulated cells divided by averagecounts/min in cells cultured without added SPE A or mutants.

Wild type SPE A was significantly superantigenic at doses from 1 to0.001 μg/well (FIG. 7). SPE A K157E was significantly mitogenic at dosesof 0.01 and 0.001 μg/well (FIG. 7). The three other SPE A mutants (SPE AN20D, SPE A N20D/C98S, SPE A N20D/K157E) were significantly lesssuperantigenic (FIG. 8) than wild type SPE A at doses of 1 to 0.001 μg(p<0.001). Interestingly, SPE A N20D was significantly moresuperantigenic (FIG. 8) than SPE A N20D/C98S at doses of 1 and 0.1 μg(p<0.0005, p<0.001, respectively). Furthermore, SPE A N20D was moremitogenic than SPE A N20D/K157E at the 1 μg/well dose (p<0.01). Thus,the data indicated the N20D/C98S mutant had less toxicity than thesingle N20D mutant, and the double mutant N20D/K157E was intermediatebetween the other two proteins. All three mutants were significantlyless toxic than wild type SPE A.

In a second experiment rabbits (3/group) were challenged iv with 10μg/kg SPE A or mutants and then endotoxin 5 μg/kg) 4 hours later.Animals were monitored for 48 hours for enhanced lethality due toadministration of SPE and endotoxin. This assay is the most sensitive invivo measure of SPE A lethal activity. As indicated in Table 6, 0/3animals challenged with wild type SPE A and endotoxin survived. Incontrast all but one animal challenged with SPE A N20D survived, and allanimals challenged with SPE A N20D/C98S or SPE A N20D/K157E survived.TABLE 6 Capacity of SPE A (10 μg/kg) or mutants (10 μg/kg) to enhancerabbit susceptibility to the lethal effects of endotoxin (5 μg/kg) SPE Aor Mutant Number Dead/Total Wild type SPE A 3/3 SPE A N20D 1/3 SPE AN20D/C98S 0/3 SPE A N20D/K157E 0/3Note:SPE A or mutants were administered iv at 0 hour and endotoxin iv at 4hours. Animals were monitored for 48 hours for lethality.

In a third experiment rabbits were immunized with SPE A N20D, SPE AN20D/C98S, OR SPE A N20D/K157E, and then challenged with wild type SPE A(10 μg/kg) and endotoxin (5 μg/kg or 25 μg/kg) as in the precedingexperiment. Control animals were not immunized but were challenged withwild type SPE A plus endotoxin. Rabbits were immunized every other weekfor two injections, with mutant proteins (50 μg/injection) emulsified inincomplete adjuvant (Freunds, Sigma Chemical Co., St. Louis, Mo.) andthen rested one week prior to challenge with wild type toxin. Thecombination of wild type SPE A and endotoxin represent 20 LD50 forchallenge with 10 μg/kg SPE A and 5 μg/kg endotoxin, and 100 LD50 forchallenge with 10 μg/kg SPE A and 25 μg/kg endotoxin.

As indicated in Table 7, all animals challenged with 100 LD50 of SPE Aand endotoxin succumbed. Similarly, all animals immunized with SPE AN20D or N20D/K157E succumbed when challenged with 20 LD50 of SPE A andendotoxin. In contrast, animals immunized with the double mutantN20D/C98S survived. Animals immunized with the double mutant N20D/K157Esuccumbed earlier than other animals. The data above indicates thatdouble mutants and in particular SPE A N20D/C98S shows effectiveness asa toxoid vaccine in test animals. TABLE 7 Ability of SPE A mutants toimmunize rabbits against the capacity of wild type SPE A to enhancesusceptibility to lethal endotoxin shock. Number Challenge dose of Dead/Immunizing Agent SPE A and Endotoxin Total None 10 μg/kg SPE A, 25 μg/kgendotoxin 3/3 SPE A N20D 10 μg/kg SPE A, 25 μg/kg endotoxin 2/2 SPE AN20D/C98S 10 μg/kg SPE A, 25 μg/kg endotoxin 2/2 SPE A N20D/K157E 10μg/kg SPE A, 25 μg/kg endotoxin 2/2 None 10 μg/kg SPE A, 5 μg/kgendotoxin 3/3 SPE A N20D 10 μg/kg SPE A, 5 μg/kg endotoxin 2/2 SPE AN20D/C98S 10 μg/kg SPE A, 5 μg/kg endotoxin 0/3 SPE A N20D/K157E 10μg/kg SPE A, 5 μg/kg endotoxin 3/3Note:Some animals escaped during this experiment. These animals were notincluded in the above data.

EXAMPLE 7 SPE A Inhibition by Antibodies to SPE-A Mutants and SPE-AMutant Immunization

One ml of blood was drawn from the marginal ear vein from each of therabbits immunized with N20D, N20D/C98S, and N20D/K157E SPE A andnonimmunized controls. Animals were bled 6 days after the lastimmunization (one 25 day before animals were used in the experiment inTable 6). After the blood clotted, sera were separated by centrifugation(13,000×g, 10 min). Sera from each group were pooled and treated with33⅓% (final concentration) of ammonium sulfate for 1 hr at roomtemperature to precipitate immunoglobulins. Precipitated immunoglobulinswere collected by centrifugation (13,000×g, 10 min), resolubilized tothe original volume in phosphate-buffered saline (0.005M NaPO4 H7.0,0.15M NaCl), and dialyzed for 24 hr against 1 liter of 0.15M NaCl at 4°C. The dialysates were filter sterilized (0.45 μm pore size) and used instudies to neutralize rabbit splenocyte mitogenicity (superantigenicity)of 0.01 μg SPE A (FIG. 9). Serum from one rabbit immunized withsublethal doses of wild type SPE A was fractionated comparably and usedas the positive control. Twenty microliters of the immunoglobulinfractions (Igs) from each group of sera were diluted ⅕ and 1/50 withcomplete RPMI 1640 mammalian cell culture media (dilution with respectto the original serum volume) and added to each of 4 wells containingwild type SPE A and 2×105 rabbit splenocytes in our standardmitogenicity assay. Igs and wild type toxin were both added tolymphocytes at time 0. The results are shown in FIG. 9.

The ⅕ diluted Igs, whether from immunized animals or nonimmune controlswere inhibitory to splenocyte proliferation, probably because ofresidual ammonium sulfate in the Igs. However, Igs from the SPE A immuneanimals and Igs from N20D, N20D/C98S, and N20D/K157E immune animals weremore inhibitory than Igs from nonimmune controls (p=0.006 for SPE Aversus nonimmune, [=0.035 for N20D versus nonimmune, p=0.0002 forN20D/C98S versus nonimmune, and p=0.0001 for N20D/K157E versus nonimmuneby use of Student's t test analysis of normally distributed unpaireddata), indicating specific inhibition of mitogenicity.

When Igs were added at the 1/50 dilution, the double mutant N20D/C98Scaused significant inhibition of splenocyte proliferation compared tononimmune controls (p=0.046). At this Ig concentration none of thefractions caused nonspecific suppression of lymphocyte mitogenicity.

These data suggest that the double mutant N20D/C98S was better able toimmunize animals against mitogenicity of the wild type SPE A than thesingle mutant N20D or the other double mutant N20D/K157E. However, thedouble mutant N20D/K157E was a better immunogen than the single mutantN20D.

Without being bound by the following, it is possible the two changes inthe N20D/C98S mutant interfere with host cell receptor sites requiredfor lethality, T cell receptor interaction, and possibly indirectly,class II MHC interaction on antigen presenting cells. Since class II MHCinteraction depends on amino acid residues in the P barrel domain(domain B) in the standard view of the toxin, we propose also that achange in this region (such as D45N) may improve the immunogenicity ofN20D/C98S even more. The basis for this hypothesis is that wild typetoxin (and possibly mutants lacking changes in the class II MHCinteraction domain) bind directly to class II MHC molecules without therequirement for normal processing by antigen presenting cells. Mutantsthat contain amino acid changes that interfere with this direct class IIMHC interaction may be more immunogenic since the mutants maybe moreeasily internalized and processed. Thus, the triple mutantN20D/C98S/D45N will be evaluated using the methods used to evaluate theother mutants.

Sera obtained from the nonimmune controls and each group of N20D,N20D/C98S, or N20D/K157E immunized rabbits were tested directly forELISA titer against wild type SPE A (L. Hudson and F. C. Hay, PracticalImmunology 2nd Ed, 1980, Blackwell Scientific Publications, Boston p237-239.) Serum from each animal was evaluated separately. The antibodytiters obtained were averaged and are shown in Table 8. Nonimmunecontrol animals as expected had very low titers of antibodies againstSPE A. In contrast all animals immunized against the mutants hadsignificant antibody titers. The animals immunized with the doublemutant N20D/K157E had the highest average titer with the other twomutants being comparable. However, the range of titers for the N20Dimmunized animals was much greater (20, 40, 160, 640, 640 for each ofthe 6 animals) than either of the double mutants. The data suggest thedouble mutants gave more consistent immunization. TABLE 8 ELISA antibodytiters of animals immunized against N20D, N20D/C98S, N20D/K157E SPE Aand nonimmune controls Immunizing Agent Average Antibody Titera RangebNone 10 <10-20   N20D SPE A 250  20-640 N20D/C98S SPE A 80 80 N20D/K157ESPE A 425 320-640a6 animals/groupbThe lowest titer detectable was 10. Titer is the reciprocal of the lastdilution that gave a positive result.

In a final experiment animals (3/group) were immunized against N20D,N20D/C98S, or N20D/K157E (50 μg/injection iv) by administering mutantprotein every other day for 5 injections and then resting the animalsfor one day. Animals were then evaluated for immunity against theability of wild type SPE A to cause fever [20 times the minimumpyrogenic dose (MPD) 4 hours after injection/kg body weight (20 MPD-4)].SPE A is one of the most potent pyrogens known with one MPD-4 in rabbitsof 0.15 μg/kg. At the 4 hr timepoint animals were injected withendotoxin (25 μg/kg) to evaluate immunity to the enhanced susceptibilityto endotoxin shock. The results are shown in Table 9.

The nonimmune animals and those immunized with N20D SPE A showed bothsignificant fever responses (0.8° C. for both groups) and enhancedsusceptibility to endotoxin (⅔ succumbed in 48 hr in both groups). Incontrast animals immunized with either double mutant were completelyprotected from fever and the enhancement phenomenon.

Collectively, all of the above data suggest both double mutants arebetter able to immunize animals against the toxic effects of SPE A thanthe single mutant. None of the mutants themselves were toxic to theanimals. The double mutant N20D/C98S was a better immunogen thanN20D/K157E, but both were effective. TABLE 9 Ability of SPE A mutantsN20D, N20D/C98S, and N20D/K157E to immunize rabbits against SPE Apyrogenicity and lethal challenge by SPE A and endotoxin. Fever ResponseImmunizing Agent Change ° C. at 4 hr Number Dead/Total None 0.8 2/3 N20DSPE A 0.8 2/3 N20D/C98S SPE A 0.0 0/3 N20D/K157E SPE A 0.1 0/3

EXAMPLE 8 Evaluation and Properties of Triple Mutant

Construction of Triple Mutant and Stability Determination.

Mutant N20D/D45N/C98S was constructed by two rounds of subcloning. Theresulting mutant speA was sequenced to ensure that in the cloningprocess the DNA fragments containing the appropriate mutations wereligated. Plasmid pMIN 164 carrying the triple mutant speA gene wastransformed in Staphylococcus aureus RN4220, and triple mutant proteinwas produced and purified as described for the other mutants.

Protein N20D/D45N/C98S was evaluated for stability. The protein waspurified from bacterial cultures in amounts comparable to the doublemutant SPE. The triple mutant protein also reacted with polyclonalantibodies specific for wild type SPE A in double immunodiffusionassays. Moreover, N20D/D45N/C98S was resistant to trypsin cleavageequally to wild type.

Proliferative Activity of Triple Mutant Protein.

Mutant N20D/D45N/C98S was evaluated for its proliferative activity inrabbit and murine splenocytes and human PBMCs. The protein was much lessactive than wild type SPE A in inducing rabbit (Table 11) and human(Table 13) cell proliferation. In murine cells the protein activity wasclose to 50% the wild type's at 100 ng/well, and even higher when the1,000 ng/well toxin dose was used (Table 12). In rabbit cellsN20D/D45N/C98S was also less active than the single mutants tested, N20Dand D45N, and as active as the double mutant N20D/C98S (Table 11).However, in the murine system, the triple mutant induced cellproliferation equally to the single mutant protein D45N and was moreactive than N20D/C98S (Table 12), whereas in human cells N20D/D45N/C98Swas also as active as D45N at 100 ng/well, but was much less active at10 ng/well (Table 13). It appeared that the introduction of the thirdmutation increased the protein activity. Perhaps, the loss of charge ofthe Asp to Asn change at position 45 had a stabilizing effect on theprotein lacking the sulfhydryl group of Cys 98. TABLE 11 Proliferativeability of triple mutant SPE A for rabbit splenocytes, compared tosingle mutants, double mutant and wild type SPE A. 100 ng/well 10ng/well 1 ng/well cpm cpm cpm Protein (10³)^(a) SD^(b) %^(c) (10³) SD %(10³) SD % SPE A 97 8 116 20 99 7 N20D 23 4 23 9 2 8 3 1.4 3 D45N 10 1.310 21 4.5 18 3.2 1.7 3 N20D/C98S 13 2 13 2.4 1.5 2 0.5 1.5 0.5N20D/D45N/ 7.6 2 8 4.4 1.3 4 1.7 0.4 2 C98S^(a)Resulting from incorporation of [³H]thymidine into DNA ofproliferating splenocytes.^(b)For quadruplicate samples.^(c)Mutant activity divided by wild-type SPE A activity at the same doseand in the same assay × 100.

TABLE 12 Proliferative ability of triple mutant SPE A on murinesplenocytes, compared to single mutants, double mutant and wild type SPEA. 1,000 ng/well 100 ng/well cpm cpm Protein (10³)^(a) SD^(b) %^(c)(10³) SD % SPE A 23 2.5 NT^(d) N20D/C98S 0.6 0.5 2.6 NT SPE A 15 3.4 521.8 D45N 9 2.4 60 24 8.4 44 N20D/D45N/C98S 12 5 80 23 1.8 46^(a)Resulting from incorporation of [³H]thymidine into DNA ofproliferating splenocytes.^(b)For quadruplicate samples.^(c)Mutant activity divided by wild-type SPE A activity at the same doseand in the same assay × 100.^(d)NT, not tested.

TABLE 13 Proliferative ability of triple mutant SPE A on human PBMCs,compared to single mutant, double mutant and wild type SPE A. 100ng/well 10 ng/well cpm cpm Protein (10³)^(a) SD^(b) %^(c) (10³) SD % SPEA 31 3 22 2 D45N 11 1.5 35 14.4 1.5 65 N20D/C98S 2.6 1 8 0.1 0.3 0.5N20D/D45N/C98S 10 0.6 32 0.9 0.3 4^(a)Resulting from incorporation of [³H]thymidine into DNA ofproliferating splenocytes.^(b)For quadruplicate samples.^(c)Mutant activity divided by wild-type SPE A activity at the same doseand in the same assay × 100.^(d)NT, not tested.Induction of IFN-γ and TNF-α Secretion.

The N20D/D45N/C98S protein was also evaluated for its ability to inducesecretion of IFN—Y and TNF-α from murine splenocytes and human PBMCs.These cytokines were measured in the supernates of cell cultures used totest proliferation (Tables 12 and 13). Supernates for cytokinedetermination were recovered after 96 hours of incubation from the cellcultures treated with the mutant or wild type SPE As at the doses of1,000 ng/well and 100 ng/well for murine and human cells, respectively.Secretion of both IFN-γ and TNF-α was affected more in human than murinecells (Table 14). This appeared to correlate with the levels of cellproliferation observed (Tables 12 and 13). However, within each species,TNF-α secretion appeared less dependent on cell proliferation (Table14). Human cells, which proliferated equally upon stimulation with D45Nor the triple mutant protein, secreted greater amounts of TNF-α whentreated with N20D/D45N/C98S. On the contrary, murine cells, whichproliferated better when treated with N20D/D45N/C98S, secreted smalleramounts of TNF-α upon stimulation with the same toxin. Very littlesecretion of cytokines was observed in supernates of cells treated withhyaluronidase, and of untreated human cells, but murine untreatedcontrol cells were considerably active in secretion of both cytokinestested. This may in part contribute to the surprisingly high levels ofIFN-γ and TNF-α in supernates of murine cells treated with either mutantprotein tested (Table 14). TABLE 14 Induction of cytokine secretion bytriple mutant SEC A in murine splenocytes and human PBMCs IFN-γ^(a)TNF-α^(b) Human^(c) Murine⁴ Human Murine Protein pg/ml^(e) % pg/ml %pg/ml % pg/ml % N20D/D45N/C98S 2880 27 2508 41 782 59 680 72 D45N 270826 3058 50 428 32 898 96 SPE A 10484 6076 1336 940 Hyaluronidase 0 0 4611 None 0 592 2 40 305^(a)IFN-γ, interferon-γ^(b)TNF-α, tumor necrosis factor-α^(c)Human PBMCs, 2 × 10⁵/well, were stimulated with 100 ng/well of wildtype or mutant toxins. Samples for each proliferation assay wereharvested at 96 hours and cytokine concentrations in the supernates weredetermined.^(d)Murine splenocytes, 5 × 10⁵/well, were stimulated with 1,000 ng/wellof wild type or mutant toxins. Samples for each proliferation assay wereharvested at 96 hours and cytokine concentrations in the supernates weredetermined.^(e)pg/ml of cytokine released upon mutant stimulation divided by pg/mlreleased upon wild type stimulation, in the same assay

Toxicity of N20D/D45N/C98S protein. Protein N20D/D45N/C98S was assayedfor its activity in enhancing endotoxin shock in American Dutch beltedrabbits. Young adult animals were injected i.v. with 5 μg/kg of bodyweight of N20D/D45N/C98S or wild type SPE A proteins. Four hours lateranimals were administered i.v. 10 μg/kg of body weight of endotoxin fromSalmonella typhimurium. Animals were monitored for symptoms of STSS anddeath for the 48 hours after the injection of endotoxin. Results areshown in Table 15. All animals administered the N20D/D45N/C98S proteinsurvived and their necroscopic examination revealed no organ damage. Onthe contrary, all animals treated with wild type SPE A died. This resultindicated that the triple mutant toxin has no detectable toxicity invivo. TABLE 15 Lethality and toxicity of triple mutant N20D/D45N/C98SSPE A in the rabbit endotoxin enhancement model Multiorgan No dead/totalProtein toxicity^(a) animals^(b) p^(c) N20D/D45N/C98S 0/9 0/3 0.05 SPE AND^(d) 3/3^(a)As judged by necroscopic examination of liver, spleen, lungs, andheart of surviving animals only. Each damaged organ of every animal isgiven one point. The sum of possible points is 3/animal. Numbers referto total damage-points/group of animals.^(b)Animals were administered intravenously 5 μg/kg of body weight ofSPE A wild type or mutant. Four hours later they were administered 10μg/kg of body weight of endotoxin from Salmonella typhimurium.^(c)Comparison of lethality caused by SPE A triple mutants withlethality of wild type.^(d)ND, not determined.

Antigenicity of D45N and N20D/D45N/C98S proteins. The proteins D45N,N20D/C98/S, N20D/D45N/C98S and the starting mutant protein N20D wereevaluated for their abilities to stimulate in animals an antibodyresponse specific for wild type SPE A. Five groups of 5 American Dutchbelted rabbits each were either untreated or treated with one of D45N,N20D, N20D/C98S, N20D/D45N/C98S. Proteins were administeredsubcutaneously in 25 μg doses for three times in IFA, every other week.Titers of anti-SPE A antibodies were determined by ELISA in seraobtained seven days after the last immunization. As shown in Table 16,animals in all but the untreated group had antibody titers significantlyhigher than the corresponding pre-immune titers. Moreover, all TABLE 16Antigenicity of purified triple mutant SPE A in rabbits compared tosingle mutants, double mutant, and wild type SPE A Pre-immune^(a)Immune^(c) Immunizing agent titer^(b) range titer range p^(d) None 1810-20  24 20-40 0.21 N20D 60  20-160 1600 1000-2000 0.003 D45N 26 10-406400 4000-8000 0.003 N20D/C98S 32 20-80 1220  100-2000 0.03N20D/D45N/C98S 16 10-40 3400 1000-8000 0.037^(a)Rabbits were bled prior to administration of the first toxin dose.^(b)Average titer of sera from five rabbits^(c)Rabbits were bled seven days after the administration of the thirdimmunizing dose.^(d)Comparison of pre-immune serum titer within each group with thetiter after immunizations by two-tailed t-test, assuming unequalvariances

TABLE 17 Significance in titer differences of sera from groups ofanimals immunized with different agents, determined by two-tailedt-test, assuming unequal variances N20D/D45N/ Immunizing Agent None C98SD45N N20D/C98S N20D <0.001 0.0656 <0.001 0.3471 N20D/C98S 0.0015 0.07480.0185 D45N <0.001 0.0656 N20D/D45N/C98S <0.001immunized groups had antibody titers significantly higher than thenon-immune control group (Table 17). Protein D45N was the mostimmunogenic, stimulating an average titer of 6,400, and with a range ofonly two serial 1:2 dilutions (Table 16). This protein was significantlymore effective as an antigen than N20D (Table 17). When D45N was presentin the same molecule as N20D and C98S its immunogenic ability decreasedconsiderably, as indicated by the average titer of 3,400 (Table 17). Theconsistency of the antibody response to N20D/D45N/C98S was also lesscompared to N20D alone, with titers ranging between 1000 and 8000 (4 1:2dilutions). However, by comparing the log of the D45N-immune andN20D/D45N/C98S-immune titers by use of the t-test, the two groups can beconsidered different only with 10% confidence (Table 17). Similarly,titers from N20D-immune and N20D/C98S-immune rabbits (1,600 and 1,220respectively), that were not significantly different from each other(Table 26 and 27), each showed a 10% confidence difference to theN20D/D45N/C98S-immune titers. In conclusion, the N20D/D45N/C98S proteinhad an intermediate ability to elicit an antibody response to SPE A.

Protective ability of N20D/D45N/C98S. The triple mutant protein wasevaluated and compared to N20D, D45N and double mutant N20D/C98S for itsability to protect animals from challenge with the wild type SPE A.Rabbits from the previous section, whose antibody titers are shown inTable 16, were challenged by use of the miniosmotic pump model. Pumpswere loaded with 500 μg (equal to 2.5 times the lethal dose) of SPE Aobtained from S. pyogenes. Animals were monitored for symptoms of STSSand death for 15 days after implantation of the miniosmotic pumps.Rectal temperatures were taken once before, and once two days afterimplantation. All animals immunized with one of the SPE A toxoidssurvived the challenge, whereas all five animals of the non-immune groupdied (Table 18). TABLE 18 Immunizing ability of double and triple mutantSPE A compared to single mutants No. with Multiorgan fever/total No.dead/total Immunizing agents toxicity^(a) animals^(b) animals^(c) p^(d)None 17/20  4/5 5/5 N20D 0/15 0/5 0/5 0.004 N20D/C98S 0/15 0/5 0/5 0.004D45N 0/15 2/5 0/5 0.004 N20D/D45N/C98S 0/15 1/5 0/5 0.004^(a)As judge by necroscopic examination of liver, spleen, lungs, andheart. Each damaged organ of each animal is given one point. The sum ofpossible points is 20 for the control group, and 15 for the treatedgroups (lungs were omitted). Fractions refer to totaldamage-points/total point per group of animals.^(b)In degrees Celsius. Rectal temperatures were taken at baseline andat day 2 after implantation of miniosmotic pumps. Fever was consideredas any temperature increment ≧0.5° C.^(c)Miniosmotic pumps were loaded with 500 μg of wild type SPE A.^(d)Comparison of lethality data of the vaccinated group of animalsversus the untreated group by Fisher's exact probability test.Lethality results were significant (p=0.004). Four animals of thenon-immune group had a significant increase (more than 0.5° C.) in bodytemperature (Table 18). Of the immunized groups, the D45N— andN20D/D45N/C98S-immune had some animals developing fever (⅖ and ⅕respectively). All animals were evaluated for gross organ abnormalitieseither after death (controls) or after being euthanized (treated). Noneof the immunized animals had any organ damage (Table 18). This indicatedthat the vaccination did not have toxic effects on the rabbit and thatthe antibodies to the toxoids in all animals were able to block toxicityof the challenging wild type SPE A. Conversely, the non-immune animalshad 17 organ damage-points out of the possible 20 (Table 18), indicatingthat each rabbit had at least two abnormal-looking organs. These resultstogether indicated that the vaccination with the N20D/D45N/C98S mutantwas safe and effective in protecting animals in an STSS model.

Although the invention has been described in the context of particularembodiments, it is intended that the scope of coverage of the patent notbe limited to those particular embodiments, but is determined byreference to the following claims.

1. A mutant SPE-A toxin or fragment thereof, wherein the mutant has atleast one amino acid change and is substantially nonlethal compared witha protein substantially corresponding to wild type SPE-A toxin.
 2. Amutant SPE-A toxin according to claim 1, wherein the mutant SPE-A toxincomprises one to six amino acid substitutions; and wherein at least oneof the substituted amino acids is positioned in N-terminal alpha helix3, in domain B beta strand 1, in domain B beta strand 2, in domain Bbeta strand 3, in domain A beta strand 6, in domain A beta strand 8, indomain A beta strand 9, in domain A beta strand 10, or is a cysteine. 3.A mutant SPE-A toxin according to claim 1, wherein the mutant SPE-Atoxin comprises one to six amino acid substitutions; and wherein atleast one of the substituted amino acids is asparagine-20, aspartic acid45, lysine-157, or cysteine-98.
 4. The mutant SPE-A toxin of claim 3,wherein the at least one amino acid substitution comprises thesubstitution of asparagine-20 to aspartic acid, glutamic acid, lysine orarginine; the substitution of cysteine 98 to serine, alanine, glycine,or threonine; the substitution of lysine-157 to glutamic acid oraspartic acid; or the substitution of aspartic acid-45 to asparagine,glutamine, serine, threonine, or alanine.
 5. The mutant SPE-A toxin ofclaim 4, wherein the at least one amino acid substitution comprisesasparagine-20 to aspartic acid, cysteine 98 to serine, aspartic acid-45to asparagine, or lysine-157 to glutamic acid.
 6. The mutant SPE-A toxinof claim 3, wherein the at least one amino acid substitution comprisessubstitution of asparagine-20.
 7. The mutant SPE-A toxin of claim 6,wherein the substitution is asparagine-20 to aspartic acid.
 8. Themutant SPE-A toxin of claim 6, further comprising substitution ofcysteine-98, or lysine-157.
 9. The mutant SPE-A toxin of claim 8,wherein the substitution is cysteine 98 to serine, or lysine-157 toglutamic acid.
 10. The mutant SPE-A toxin of claim 6, further comprisingsubstitution of cysteine-98 and aspartic acid-45.
 11. The mutant SPE-Atoxin of claim 10, wherein the cysteine-98 is substituted to serine andaspartic acid-45 is substituted to asparagine.
 12. The mutant SPE-Atoxin of claim 1, wherein the mutant has at least one of the followingcharacteristics: the mutant has a decrease in mitogenicity for T-cells,the mutant does not substantially enhance endotoxin shock, the mutant isnot lethal, or the mutant is nonlethal but retains mitogenicitycomparable to that of the wild type SPE-A toxin.
 13. A vaccine forprotecting animals against at least one biological activity of wild-typeSPE-A comprising: an effective amount of at least one mutant SPE-A toxinaccording to claim
 1. 14. A pharmaceutical composition comprising: amutant SPE-A according to claim 1 in admixture with a physiologicallyacceptable carrier.
 15. A DNA sequence encoding a mutant SPE-A toxinaccording to claim
 1. 16. A stably transformed host cell comprising aDNA sequence according to claim
 15. 17. A method for protecting ananimal against at least one biological activity of a wild type SPE-Acomprising: administering a vaccine according to claim 13 to an animal.18. A method for reducing symptoms associated with toxic shockcomprising: administering a vaccine according to claim 13 to an animal.